Compounds, compositions, and methods for the treatment of synucleinopathies

ABSTRACT

Bis- and tris-dihydroxyaryl compounds and their methylenedioxy analogs and pharmaceutically acceptable esters, their synthesis, pharmaceutical compositions containing them, and their use in the treatment of synucleinopathies, such as Parkinson&#39;s disease, and the manufacture of medicaments for such treatment.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/800,260, filed on May 4, 2007 now abandoned, which is a continuationin part of U.S. application Ser. No. 10/452,851 filed May 30, 2003 nowU.S. Pat. No. 7,514,583 which claimed priority under 35 USC 119(e) to:

-   (1) U.S. Provisional Application No. 60/385,144, filed May 31, 2002,-   (2) U.S. Provisional Application No. 60/409,100, filed Sep. 9, 2002,-   (3) U.S. Provisional Application No. 60/412,272, filed Sep. 20,    2002,-   (4) U.S. Provisional Application No. 60/435,880, filed Dec. 20,    2002, and-   (5) U.S. Provisional Application No. 60/463,104, filed Apr. 14,    2003.    The entire contents of all of these applications are incorporated by    reference into this application.

TECHNICAL FIELD

This invention relates to bis- and tris-dihydroxyaryl compounds andtheir methylenedioxy analogs and pharmaceutically acceptable esters,their synthesis, pharmaceutical compositions containing them, and theiruse in the treatment of amyloid diseases, especially Aβ amyloid disease,such as observed in Alzheimer's disease, IAPP amyloid disease, such asobserved in type 2 diabetes, and synucleinopathies, such as observed inParkinson's disease, and in the manufacture of medicaments for suchtreatment.

BACKGROUND OF THE INVENTION

Alzheimer's disease is characterized by the accumulation of a 39-43amino acid peptide termed the β-amyloid protein or Aβ, in a fibrillarform, existing as extracellular amyloid plaques and as amyloid withinthe walls of cerebral blood vessels. Fibrillar Aβ amyloid deposition inAlzheimer's disease is believed to be detrimental to the patient andeventually leads to toxicity and neuronal cell death, characteristichallmarks of Alzheimer's disease. Accumulating evidence implicatesamyloid, and more specifically, the formation, deposition, accumulationand/or persistence of Aβ fibrils, as a major causative factor ofAlzheimer's disease pathogenesis. In addition, besides Alzheimer'sdisease, a number of other neurodegenerative amyloid diseases involveformation, deposition, accumulation and persistence of Aβ fibrils,including Down's syndrome, disorders involving congophilic angiopathy,such as but not limited to, hereditary cerebral hemorrhage of the Dutchtype, inclusion body myositosis, dementia pugilistica, cerebralβ-amyloid angiopathy, dementia associated with progressive supranuclearpalsy, dementia associated with cortical basal degeneration and mildcognitive impairment.

Parkinson's disease is another neurodegenerative human disordercharacterized by the formation, deposition, accumulation and/orpersistence of abnormal fibrillar protein deposits that demonstrate manyof the characteristics of amyloid. In Parkinson's disease, anaccumulation of cytoplasmic Lewy bodies consisting of filaments ofα-synuclein/NAC (non-Aβ component) are believed important in thepathogenesis and as therapeutic targets. New agents or compounds able toinhibit α-synuclein and/or NAC formation, deposition, accumulationand/or persistence, or disrupt pre-formed α-synuclein/NAC fibrils (orportions thereof) are regarded as potential therapeutics for thetreatment of Parkinson's and related synucleinopathies. NAC is a 35amino acid fragment of α-synuclein that has the ability to formamyloid-like fibrils either in vivo or as observed in the brains ofpatients with Parkinson's disease. The NAC fragment of α-synuclein is arelative important therapeutic target as this portion of α-synuclein isbelieved crucial for formation of Lewy bodies as observed in allpatients with Parkinson's disease, synucleinopathies and relateddisorders.

A variety of other human diseases also demonstrate amyloid depositionand usually involve systemic organs (i.e. organs or tissues lyingoutside the central nervous system), with the amyloid accumulationleading to organ dysfunction or failure. These amyloid diseases(discussed below) leading to marked amyloid accumulation in a number ofdifferent organs and tissues, are known as systemic amyloidoses. Inother amyloid diseases, single organs may be affected such as thepancreas in 90% of patients with type 2 diabetes. In this type ofamyloid disease, the beta-cells in the islets of Langerhans in pancreasare believed to be destroyed by the accumulation of fibrillar amyloiddeposits consisting primarily of a protein known as islet amyloidpolypeptide (IAPP). Inhibiting or reducing such IAPP amyloid fibrilformation, deposition, accumulation and persistence is believed to leadto new effective treatments for type 2 diabetes. In Alzheimer's disease,Parkinson's and “systemic” amyloid diseases, there is currently no cureor effective treatment, and the patient usually dies within 3 to 10years from disease onset.

The amyloid diseases (amyloidoses) are classified according to the typeof amyloid protein present as well as the underlying disease. Amyloiddiseases have a number of common characteristics including each amyloidconsisting of a unique type of amyloid protein. The amyloid diseasesinclude, but are not limited to, the amyloid associated with Alzheimer'sdisease, Down's syndrome, hereditary cerebral hemorrhage withamyloidosis of the Dutch type, dementia pugilistica, inclusion bodymyositosis (Askanas et al, Ann. Neurol. 43:521-560, 1993) and mildcognitive impairment (where the specific amyloid is referred to asbeta-amyloid protein or Aβ), the amyloid associated with chronicinflammation, various forms of malignancy and Familial MediterraneanFever (where the specific amyloid is referred to as AA amyloid orinflammation-associated amyloidosis), the amyloid associated withmultiple myeloma and other B-cell dyscrasias (where the specific amyloidis referred to as AL amyloid), the amyloid associated with type 2diabetes (where the specific amyloid protein is referred to as amylin orislet amyloid polypeptide or IAPP), the amyloid associated with theprion diseases including Creutzfeldt-Jakob disease, Gerstmann-Strausslersyndrome, kuru and animal scrapie (where the specific amyloid isreferred to as PrP amyloid), the amyloid associated with long-termhemodialysis and carpal tunnel syndrome (where the specific amyloid isreferred to as α₂-microglobulin amyloid), the amyloid associated withsenile cardiac amyloidosis and Familial Amyloidotic Polyneuropathy(where the specific amyloid is referred to as transthyretin orprealbumin), and the amyloid associated with endocrine tumors such asmedullary carcinoma of the thyroid (where the specific amyloid isreferred to as variants of procalcitonin). In addition, the α-synucleinprotein which forms amyloid-like fibrils, and is Congo red andThioflavin S positive (specific stains used to detect amyloid fibrillardeposits), is found as part of Lewy bodies in the brains of patientswith Parkinson's disease, Lewy body disease (Lewy in Handbuch derNeurologie, M. Lewandowski, ed., Springer, Berlin pp. 920-933, 1912;Pollanen et al., J. Neuropath. Exp. Neurol. 52:183-191, 1993;Spillantini et al, Proc Natl. Acad. Sci. USA 95:6469-6473, 1998; Arai etal, Neuropath. Lett. 259:83-86, 1999), multiple system atrophy(Wakabayashi et al, Acta Neuropath. 96:445-452, 1998), dementia withLewy bodies, and the Lewy body variant of Alzheimer's disease. Forpurposes of this disclosure, Parkinson's disease, due to the fact thatfibrils develop in the brains of patients with this disease (which areCongo red and Thioflavin S positive, and which contain predominantbeta-pleated sheet secondary structure), is now regarded as a diseasethat also displays the characteristics of an amyloid-like disease.

Systemic amyloidoses which include the amyloid associated with chronicinflammation, various forms of malignancy and familial Mediterraneanfever (i.e. AA amyloid or inflammation-associated amyloidosis) (Bensonand Cohen, Arth. Rheum. 22:36-42, 1979; Kamei et al, Acta Path. Jpn32:123-133, 1982; McAdam et al., Lancet 2:572-573, 1975; Metaxas, KidneyInt. 20:676-685, 1981), and the amyloid associated with multiple myelomaand other B-cell dyscrasias (i.e. AL amyloid) (Harada et al., J.Histochem Cytochem 19:1-15, 1971), as examples, are known to involveamyloid deposition in a variety of different organs and tissuesgenerally lying outside the central nervous system. Amyloid depositionin these diseases may occur, for example, in liver, heart, spleen,gastrointestinal tract, kidney, skin, and/or lungs (Johnson et al, N.Engl. J. Med. 321:513-518,1989). For most of these amyloidoses, there isno apparent cure or effective treatment and the consequences of amyloiddeposition can be detrimental to the patient. For example, amyloiddeposition in the kidney may lead to renal failure, whereas amyloiddeposition in the heart may lead to heart failure. For these patients,amyloid accumulation in systemic organs leads to eventual deathgenerally-within 3-5 years. Other amyloidoses may affect a single organor tissue such as observed with the Aβ amyloid deposits found in thebrains of patients with Alzheimer's disease and Down's syndrome: the PrPamyloid deposits found in the brains of patients with Creutzfeldt-Jakobdisease, Gerstmann-Straussler syndrome, and kuru; the islet amyloid(IAPP) deposits found in the islets of Langerhans in the pancreas of 90%of patients with type 2 diabetes (Johnson et al, N. Engl. J. Med321:513-518, 1989; Lab. Invest. 66:522 535, 1992); the α-microglobulinamyloid deposits in the medial nerve leading to carpal tunnel syndromeas observed in patients undergoing long-term hemodialysis (Geyjo et al,Biochem Biophys. Res. Comm. 129:701-706, 1985; Kidney Int. 30:385-390,1986); the prealbumin/transthyretin amyloid observed in the hearts ofpatients with senile cardiac amyloid; and the prealbumin/transthyretinamyloid observed in peripheral nerves of patients who have familialamyloidotic polyneuropathy (Skinner and Cohen, Biochem Biophys. Res.Comm. 99:1326-1332, 1981; Saraiva et al, J. Lab. Clin. Med. 102:590-603,1983; J. Clin. Invest. 74:104-119, 1984; Tawara et al, J. Lab. Clin.Med. 98:811-822, 1989).

Alzheimer's disease also puts a heavy economic burden on society Arecent study estimated that the cost of caring for one Alzheimer'sdisease patient with severe cognitive impairments at home or in anursing home, is more than $47,000 per year (A Guide to UnderstandingAlzheimer's Disease and Related Disorders). For a disease that can spanfrom 2 to 20 years, the overall cost of Alzheimer's disease to familiesand to society is staggering. The annual economic toll of Alzheimer'sdisease in the United States in terms of health care expenses and lostwages of both patients and their caregivers is estimated at $80 to $100billion (2003 Progress Report on Alzheimers Disease).

Tacrine hydrochloride (“Cognex”), the first FDA approved drug forAlzheimer's disease, is a acetylcholinesterase inhibitor (Cutler andSramek, N. Engl. J. Med. 328:808 810, 1993). However, this drug hasshowed limited success in producing cognitive improvement in Alzheimer'sdisease patients and initially had major side effects such as livertoxicity The second FDA approved drug, donepezil (“Aricept”), which isalso an acetylcholinesterase inhibitor, is more effective than tacrine,by demonstrating slight cognitive improvement in Alzheimer's diseasepatients (Barner and Gray, Ann. Pharmacotherapy 32:70-77, 1998; Rogersand Friedhoff, Eur. Neuropsych. 8:67-75, 1998), but is not believed tobe a cure. Therefore, it is clear that there is a need for moreeffective treatments for Alzheimer's disease patients.

Amyloid as a Therapeutic Target for Alzheimer's Disease

Alzheimer's disease is characterized by the deposition and accumulationof a 39-43 amino acid peptide termed the beta-amyloid protein, Aβ orβ/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984;Masters et al., Proc. Natl. Acid Sci. USA 82:4245-4249, 1985; Husby etal., Bull. WHO 71:105-108, 1993). Aβ is derived by protease cleavagefrom larger precursor proteins termed β-amyloid precursor proteins(APPs) of which there are several alternatively spliced variants. Themost abundant forms of the APPs include proteins consisting of 695, 751and 770 amino acids (Tanzi et al., Nature 31:528-530, 1988).

The small Aβ peptide is a major component that makes up the amyloiddeposits of “plaques” in the brains of patients with Alzheimer'sdisease. In addition, Alzheimer's disease is characterized by thepresence of numerous neurofibrillary “tangles”, consisting of pairedhelical filaments which abnormally accumulate in the neuronal cytoplasm(Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986;Kosik et al., Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al.,Science 251:675-678, 1991). The pathological hallmark of Alzheimer'sdisease is therefore the presence of “plaques” and “tangles”, withamyloid being deposited in the central core of the plaques. The othermajor type of lesion found in the Alzheimer's disease brain is theaccumulation of amyloid in the walls of blood vessels, both within thebrain parenchyma and in the walls of meningeal vessels that lie outsidethe brain. The amyloid deposits localized to the walls of blood vesselsare referred to as cerebrovascular amyloid or congophilic angiopathy(Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986; Pardridge et al.,J. Neurochem. 49:1394-1401, 1987)

For many years there has been an ongoing scientific debate as to theimportance of “amyloid” in Alzheimer's disease, and whether the“plaques” and “tangles” characteristic of this disease were a cause ormerely a consequence of the disease. Within the last few years, studiesnow indicate that amyloid is indeed a causative factor for Alzheimer'sdisease and should not be regarded as merely an innocent bystander. TheAlzheimer's Aβ protein in cell culture has been shown to causedegeneration of nerve cells within short periods of time (Pike et al.,Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1995). Studiessuggest that it is the fibrillar structure (consisting of a predominantβ-pleated sheet secondary structure), characteristic of all amyloids,that is responsible for the neurotoxic effects. Aβ has also been foundto be neurotoxic in slice cultures of hippocampus (Harrigan et al.,Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death intransgenic mice (Games et al., Nature 373:523-527, 1995; Hsiao et al.,Science 274:99-102, 1996). Injection of the Alzheimer's Aβ into ratbrain also causes memory impairment and neuronal dysfunction (Flood etal., Proc. Natl. Acad. Sci. USA 88:3363-3366, 1991; Br. Res.663:271-276, 1994).

Probably, the most convincing evidence that Aβ amyloid is directlyinvolved in the pathogenesis of Alzheimer's disease comes from geneticstudies. It was discovered that the production of Aβ can result frommutations in the gene encoding, its precursor, β-amyloid precursorprotein (Van Broeckhoven et al., Science 248:1120-1122, 1990; Murrell etal., Science 254:97-99, 1991; Hsiao et al., Nature Med. 1:1291-1296,1995). The identification of mutations in the beta-amyloid precursorprotein gene that cause early onset familial Alzheimer's disease is thestrongest argument that amyloid is central to the pathogenetic processunderlying this disease. Four reported disease-causing mutations havebeen discovered which demonstrate the importance of Aβ in causingfamilial Alzheimer's disease (reviewed in Hardy, Nature Genet.1:233-234, 1992). All of these studies suggest that providing a drug toreduce, eliminate or prevent fibrillar Aβ formation, deposition,accumulation and/or persistence in the brains of human patients willserve as an effective therapeutic.

Parkinson's Disease and Synucleinopathies

Parkinson's disease is a neurodegenerative disorder that ispathologically characterized by the presence of intracytoplasmic Lewybodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer,Berlin, pp. 920-933, 1912; Pollanen et al., J. Neuropath. Exp. Neurol.52:183-191, 1993), the major components of which are filamentsconsisting of α-synuclein (Spillantini et al., Proc. Natl. Acad. Sci.USA 95:6469-6473, 1998; Arai et al., Neurosci. Lett. 259:83-86, 1999), a140-amino acid protein (Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993). Two dominant mutations in α-synuclein causingfamilial early onset Parkinson's disease have been described suggestingthat Lewy bodies contribute mechanistically to the degeneration ofneurons in Parkinson's disease and related disorders (Polymeropoulos etal., Science 276:2045-2047, 1997; Kruger et al., Nature Genet.18:106-108, 1998). Recently, in titro studies have demonstrated thatrecombinant α-synuclein can indeed form Lewy body-like fibrils (Conwayet al., Nature Med. 4:1318-1320, 1998; Hashimoto et al., Brain Res.799:301-306, 1998; Nahri et al, J. Biol. Chem. 274:9843-9846, 1999).Most importantly, both Parkinson's disease-linked α-synuclein mutationsaccelerate this aggregation process, demonstrating that such in vitrostudies may have relevance for Parkinson's disease pathogenesis.Alpha-synuclein aggregation and fibril formation fulfills of thecriteria of a nucleation-dependent polymerization process (Wood et al.,J. Biol. Chem. 274:19509-19512, 1999). In this regard α-synuclein fibrilformation resembles that of Alzheimer's β-amyloid protein (Aβ) fibrils.Alpha-synuclein recombinant protein, and non-Aβ component (known asNAC), which is a 35-amino acid peptide fragment of α-synuclein, bothhave the ability to form fibrils when incubated at 37° C., and arepositive with amyloid stains such as Congo red (demonstrating ared/green birefringence when viewed under polarized light) andThioflavin S (demonstrating positive fluorescence) (Hashimoto et al.,Brain Res. 799:301-306, 1998; Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993).

Synucleins are a family of small, presynaptic neuronal proteins composedof α-, β-, and γ-synucleins, of which only α-synuclein aggregates havebeen associated with several neurological diseases (Ian et al., ClinicalNeurosc. Res. 1:445-455, 2001; Trojanowski and Lee, Neurotoxicology23:457-460, 2002). The role of synucleins (and in particular,alpha-synuclein) in the etiology of a number of neurodegenerative and/oramyloid diseases has developed from several observations.Pathologically, synuclein was identified as a major component of Lewybodies, the hallmark inclusions of Parkinson's disease, and a fragmentthereof was isolated from amyloid plaques of a different neurologicaldisease, Alzheimer's disease. Biochemically, recombinant α-synuclein wasshown to form amyloid-like fibrils that recapitulated theultrastructural features of alpha-synuclein isolated from patients withdementia with Lewy bodies, Parkinson's disease and multiple systematrophy. Additionally, the identification of mutations within thesynuclein gene, albeit in rare cases of familial Parkinson's disease,demonstrated an unequivocal link between synuclein pathology andneurodegenerative diseases. The common involvement of α-synuclein in aspectrum of diseases such as Parkinson's disease, dementia with Lewybodies, multiple system atrophy and the Lewy body variant of Alzheimer'sdisease has led to the classification of these diseases under theumbrella term of “synucleinopathies”.

Parkinson's disease α-synuclein fibrils, like the Aβ fibrils ofAlzheimer's disease, also consist of a predominantly β-pleated sheetstructure. Therefore, compounds found to inhibit Alzheimer's disease Aβamyloid fibril formation are also anticipated to be effective in theinhibition of α-synuclein/NAC fibril formation, as shown from Examplesin the present invention. These compounds would therefore also serve astherapeutics for Parkinson's disease and other synucleinopathies, inaddition to having efficacy as a therapeutic for Alzheimer's disease,type 2 diabetes, and other amyloid disorders.

Parkinson's disease and Alzheimer's disease are characterized by theinappropriate accumulation of insoluble aggregates comprised primarilyof misfolded proteins that are enriched in β-pleated sheet secondarystructure (reviewed in Cohen et al., Nature 426:905-909, 2003; Chiti etal., Annu. Rev. Biochem., 75:333-366, 2006). In Parkinson's disease,α-synuclein is the major constituent of these aggregates, as part ofLewy Bodies, and mutations in α-synuclein that increase its propensityto misfold and aggregate are observed in familial Parkinson's disease(Polymeropoulos et al., Science 276:1197-1199, 1997; Papadimitriou etal., Neurology 52:651-654, 1999).

Mitochondrial dysfunction, specifically as a result of impairment atcomplex I of the electron transport chain, is also a common feature ofParkinson's disease (Schapira et al., J. Neurochem., 54:823-827, 1990;reviewed in Greenamyre et al., IUBMB Life., 52:135-141, 2001). Directevidence for mitochondrial deficits in the etiology of Parkinson'sdisease came first from the observation thatMPP+(1-methyl-4-phenyl-2,3-dihydropridinium), the active metabolite ofthe parkinsonism toxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), inhibits complex I (Nicklas et al., L. Sci, 36:2503-2508, 1985).Subsequently, rotenone, another complex I inhibitor, was shown to be animproved model for α-synuclein aggregation because it reproduces theabove-mentioned α-synuclein-positive intracytoplasmic aggregates, inaddition to the behavioral changes and loss of dopaminergic neurons seenin the MPTP model. Rotenone toxicity of this type is seen in multiplemodel systems including rats (Betarbet et al., Nat Neurosci.3:1301-1306, 2000; Panov et al., J. Bio. Chem., 280:42026-42035, 2005),rat brain slices (Sherer et al., J. Neurosci., 23:10756-10764, 2003;Testa et al., Mol. Brain Res., 134:109-118, 2005), C. elegans (Ved etal., J. Biol. Chem, 280:42655-42668, 2005) and cultured cells (Sherer etal., J. Neurosci, 22:7006-7015, 2002) and has been shown to be aconsequence of increased oxidative damage resulting from complex Iinhibition.

To better understand the relationship of oxidative damage to mutantα-synuclein pathogenesis, a neuroblastoma cell line (using BE-M17 cells)has been established in the art that overexpresses A53T α-synuclein. Inthese cells, A53T α-synuclein aggregates in response to a variety ofoxidative stress-inducing agents and potentiates mitochondrialdysfunction and cell death (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000). These cells are also amenable to rotenone treatmentand hence, are particularly useful for testing agents that might inhibitα-synuclein aggregation/fibrillogenesis.

Discovery and identification of new compounds or agents as potentialtherapeutics to arrest amyloid formation, deposition, accumulationand/or persistence that occurs in Alzheimer's disease, Parkinson'sdisease, type II diabetes, and other amyloidoses are desperately sought.

SUMMARY OF THE INVENTION

In a first aspect, this invention is bis- and tris-dihydroxyarylcompounds and their methylenedioxy analogs and pharmaceuticallyacceptable esters, and pharmaceutically acceptable salts thereof. Thecompounds are useful in the treatment of amyloid diseases andsynucleinopathies.

The compounds are:

-   (1) compounds of the formula:

where:

-   R is a C₁-C₁₀ alkylene group, in which, when the number of carbon    atoms is at least 2, there are optionally 1 or 2 non-adjacent double    bonds; 1 to 3 non-adjacent methylene groups are optionally replaced    by NR′ (where R′ is H, alkyl, or acyl), O, or S; and 1 or 2    methylene groups are optionally replaced by a carbonyl or    hydroxymethylene group; and-   (2) the compounds that are:-   3,4,3′,4′-tetrahydroxybenzoin (compound 1);    3,4,3′,4′-tetrahydroxydesoxybenzoin (compound 2);    3,4,3′,4′-tetrahydroxydiphenylmethane (compound 3);    1,2-bis(3,4-dihydroxyphenyl)ethane (compound 4);    1,3-bis(3,4-dihydroxyphenyl)propane (compound 5);    3,4,3′,4′-tetrahydroxychalcone (compound 6);    3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline (compound 7);    4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine (compound 8);    1,4-bis(3,4-dihydroxybenzyl)piperazine (compound 9);-   N,N′-bis(3,4-dihydroxybenzyl)-N,N′-dimethylethylenediamine (compound    10); 2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane    (compound 11);    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane (compound    12); N,N′-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane    (compound 13);    N,N′-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane    (compound 14); N-(3,4-dihydroxybenzyl)proline    3,4-dihydroxybenzylamide (compound 15);    2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid    3,4-dihydroxyphenethylamide (compound 16);    2,6-bis(3,4-dihydroxybenzyl)cyclohexanone (compound 17);    3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone (compound 18);    2,4-bis(3,4-dihydroxybenzyl)-3-tropinone (compound 19);    tris-(3,4-dihydroxybenzy) methane (compound 20);    α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid    3,4-dihydroxybenzyl amide (compound 21);    4-(3,4-dihydroxybenzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one    (compound 22); 1,4-bis(3,4-dihydroxybenzoyl)piperazine (compound    23); N,N′-bis(3,4-dihydroxybenzoyl)-N,N′-dimethylethylenediamine    (compound 24);    2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane    (compound 25);    N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane    (compound 26);    N,N′-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane    (compound 27); 3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine    (compound 28);    3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine    (compound 29); N-(3,4-dihydroxyphenylacetyl)proline    3,4-dihydroxyanilide (compound 30);    2,3-bis(3,4-dihydroxypheny)butane (compound 31);    1,3-bis(3,4-dihydroxybenzyl)benzene (compound 32);    1,4-bis(3,4-dihydroxybenzyl)benzene (compound 33);    2,6-bis(3,4-dihydroxybenzyl)pyridine (compound 34);    2,5-bis(3,4-dihydroxybenzyl)thiophene (compound 35);    2,3-bis(3,4-dihydroxybenzyl)thiophene (compound 36);    1,2-bis(3,4-dihydroxyphenyl)-cyclohexane (compound 37);    1,4-bis(3,4-dihydroxyphenyl)cyclohexane (compound 38);    3,7-bis(3,4-dihydroxyphenyl)bicyclo[3.3.0]octane (compound 39);    2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethylbicyclo[2.2.1]heptane    (compound 40); 1,2-bis(3,4-dihydroxyphenoxy)ethane (compound 41);-   1,3-bis(3,4-dihydroxy)phenoxy)propane (compound 42);    trans-1,2-bis(3,4-dihydroxyphenoxy)-cyclopentane (compound 43);    N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine    (compound 44); 3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide    (compound 45); 3,4-dihydroxyphenoxyacetic acid    3,4-dihydroxybenzylamide (compound 46); 3,4-dihydroxyphenoxyacetic    acid 3,4-dihydroxyphenethylamide (compound 47); 3,4-dihydroxybenzoic    acid p-(3,4-dihydroxyphenoxy)anilide (compound 48);    3,4-dihydroxybenzoic acid o-(3,4-dihydroxyphenoxy)anilide (compound    49); 2,6-bis(3,4-dihydroxyphenoxy)pyridine (compound 50),    3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compound 51);    3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide (compound 52);    3,4-dihydroxybenzoic acid 3,4-dihydroxyphenethylamide (compound 53);    3,4-dihydroxyphenylacetic acid 3,4-dihydroxyanilide (compound 54);    3,4-dihydroxyphenylacetic acid 3,4-dihydroxybenzylamide (compound    55); 3,4-dihydroxyphenylacetic acid 3,4-dihydroxyphenethylamide    (compound 56); 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxyanilide (compound 57); 3-(3,4-dihydroxyphenyl)    propionic acid 3,4-dihydroxybenzylamide (compound 58);    3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyphenethylamide    (compound 59); 3,4-dihydroxycinnamic acid 3,4-dihydroxyanilide    (compound 60); 3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide    (compound 61); 3,4-dihydroxycinnamic acid    3,4-dihydroxyphenethylamide (compound 62); oxalic acid    bis(3,4-dihydroxyanilide) (compound 63); oxalic acid    bis(3,4-dihydroxybenzylamide) (compound 64); oxalic acid    bis(3,4-dihydroxyphenethylamide) (compound 65); succinic acid    bis(3,4-dihydroxyanilide) (compound 66); succinic acid    bis(3,4-dihydroxybenzylamide) (compound 67); succinic acid    bis(3,4-dihydroxyphenethylamide) (compound 68); maleic acid    bis(3,4-dihydroxyanilide) (compound 69); maleic acid    bis(3,4-dihydroxybenzylamide) (compound 70); fumaric acid    bis(3,4-dihydroxyanilide)(compound 71); fumaric acid    bis(3,4-dihydroxybenzylamide) (compound 72);    bis(3,4-dihydroxybenzyl)amine (compound 73);    N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine (compound 74);    tris(3,4-dihydroxybenzyl)amine (compound 75);    1,3-bis(3,4-dihydroxyphenyl)urea (compound 76);    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea (compound 77);    1- (3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea (compound    78); 3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin (compound 79);    3-deoxy-3-(3,4-dihydroxyphenethyl)aminoepicatechin (compound 80);    2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine (compound 81);    10-aminoanthracene-1,2,7,8-tetraol (compound 82);    acridine-1,2,6,7-tetraol (compound 83);    phenoxazine-2,3,7,8,10-pentaol (compound 84);    dibenzo[c,f][2,7]napthyridine-2,3,10,11-tetraol (compound 85); and    6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-2,10,11-triol    (compound 86);-   (3) the methylenedioxy analogs and pharmaceutically acceptable    esters of compounds of (1) and (2); and-   (4) the pharmaceutically acceptable salts of the compounds of (1) to    (3).

In a second aspect, this invention is pharmaceutical compositionscomprising a compound of the first aspect of this invention and apharmaceutically acceptable excipient; and pharmaceutical compositionscomprising a pharmaceutically acceptable excipient and, as the soleactive ingredient, a compound of the first aspect of the invention.

In a third aspect, this invention is a method of treating an amyloiddisease or synucleinopathy in a mammal, especially a human, byadministration of a therapeutically effective amount of a compound ofthe first aspect of this invention, for example as a pharmaceuticalcomposition.

In a fourth aspect, this invention is the use of a compound of the firstaspect of this invention in the manufacture of a medicament for thetreatment of an amyloid disease or synucleinopathy.

In a fifth aspect, this invention is a method of preparation of the bis-and tris(dihydroxy-aryl) compounds of the first aspect of thisinvention, i.e. the compounds of the formula or list above, exceptcompound #86, and of their pharmaceutically acceptable esters, bydeprotection of the methylenedioxy analogs of the compounds, optionallyfollowed by the esterification of the resulting bis- andtris(dihydroxyaryl) compounds and/or the formation of pharmaceuticallyacceptable salts thereof.

In a sixth aspect, this invention is a method of treatment of Aβ, IAPP,other amyloids, and α-synuclein or NAC fibrillogenesis, in an in vitroenvironment. The method includes the step of administering into the invitro environment a therapeutically effective amount of a compound ofthis invention. Preferably the compound is selected from the groupsdescribed below with respect to their activity against Aβ, IAPP, andNAC.

In another aspect, this invention is a method of treating the formation,deposition, accumulation, or persistence of α-synuclein/NAC fibrils,comprising treating the fibrils with an effective amount of thecompounds of this invention.

In another aspect, this invention is a method of treating synucleindisease comprising administration of a therapeutically effective amountof the compounds of this invention.

In another aspect, this invention is a method resulting inneuroprotection from a synuclein disease in a mammal comprising the stepof administrating a therapeutically effective amount of the compounds ofthis invention.

In another aspect, this invention is a method resulting inneuroprotection against degenerative changes in mammals with synucleindisease comprising the step of administrating a therapeuticallyeffective amount of the compounds of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows four circular dichroism spectra showing examples ofAlzheimer's disease Aβ fibril disruption by compounds 4, 12, 51 and 61.

FIG. 2 is a circular dichroism spectrum showing an example ofAlzheimer's disease Aβ fibril disruption by compound 78.

FIG. 3 are three circular dichroism spectra showing examples ofAlzheimer's disease Aβ fibril disruption (in a dose-dependent manner) bycompounds 12, 51 and 61.

FIGS. 4A-D are examples of fluorescent photomicrographs demonstratingthe rotenone dose-dependent increase in thioflavin S-positive aggregates(green fluorescence)in the cytoplasm of BE-M17 human neuroblastoma cellsoverexpressing A53T α-synuclein. FIG. 4E summarizes the quantitativeanalysis of the rotenone dose response.

FIGS. 5A-C are examples of fluorescent photomicrographs demonstratingthat compound 51 reduces the presence of rotenone-induced thioflavinS-positive aggregates (green) in cells in a dose dependent manner. FIG.5D summarizes the quantitative analysis of the anti-aggregation effectsof compound 51.

FIGS. 6A-C are examples of fluorescent photomicrographs demonstratingthat compound 76 reduces the presence of rotenone-induced thioflavinS-positive aggregates (green) in cells in a dose dependent manner. FIG.6D summarizes the quantitative analysis of the anti-aggregation effectsof compound 76.

FIGS. 7A-B are examples of fluorescent photomicrographs demonstratingthat compound 21 reduces the presence of rotenone-induced thioflavinS-positive aggregates (green) in cells. FIG. 7C summarizes thequantitative analysis of the anti-aggregation effects of compound 21.

FIGS. 8A-C are examples of fluorescent photomicrographs demonstratingthat compound 3 reduces the presence of rotenone-induced thioflavinS-positive aggregates (green) in cells in a dose dependent manner. FIG.8D summarizes the quantitative analysis of the anti-aggregation effectsof compound 3.

FIG. 9 is a graph showing the ability of compound 51 to inhibitrotenone-induced toxicity in the XTT Cytotoxicity assay.

FIG. 10 is a graph showing the ability of compound 76 to inhibitrotenone-induced toxicity in the XTT Cytotoxicity assay.

FIG. 11 is a graph showing the ability of compound 21 to inhibitrotenone-induced toxicity in the XTT Cytotoxicity assay.

FIG. 12 is a graph showing the ability of compound 63 to inhibitrotenone-induced toxicity in the XTT Cytotoxicity assay.

FIG. 13A is an example of circular dichroism spectra illustrating theability of compound 51 to prevent the conversion or accumulation ofalpha-synuclein peptide into a n-sheet rich structure. FIG. 13B is agraph summarizing the ability of compound 51 to inhibitconversion/accumulation and halt progression of alpha-synuclein into aβ-sheet rich structure(s). FIG. 13C is a graph summarizing the compound51 dose response in inhibiting conversion or accumulation ofalpha-synuclein into a β-sheet rich structure. FIG. 13D is a graphsummarizing the ability of several compounds to inhibit the conversionor accumulation of alpha-synuclein into a β-sheet rich structure.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In this application, the following terms shall have the followingmeanings, without regard to whether the terms are used variantlyelsewhere in the literature or otherwise in the known art. The compoundsof the invention, i.e. the compounds of the formula shown in theparagraph numbered (1) at the top of page 7 of the application and thecompounds on the list immediately following and numbered (2), arereferred to generally as bis- and tris-dihydroxyaryl compounds, orsometimes just as “dihydroxyaryl compounds”. It will be noted thatcompound #84 has an additional hydroxy group, but does have twodihydroxyaryl groups; while compound #86 has only one dihydroxyarylgroup but has an additional phenolic hydroxyl moiety.

“Methylenedioxy analogs” refers to the compounds of this invention inwhich each of the pairs of adjacent hydroxyl moieties of thedihydroxyaryl groups have been replaced by methylenedioxy groups. Themethylenedioxy compounds are illustrated and referred to as compounds#1B to #86B or DC-0001B to DC-0086B. The methylenedioxy groups also areconvenient intermediate protecting groups for the dihydroxy moieties andtherefore these disclosed compounds are believed to also serve aseffective prodrugs. The methylenedioxy analogs #1B to #80B areillustrated in Example 30.

“Pharmaceutically acceptable esters” refers to the compounds of thisinvention where the hydroxyl moieties of the dihydroxyaryl groups of thecompounds are esterified with an acid or acids that result in apharmaceutically acceptable poly(ester). The compounds are shown inExample 31 as acetylated, and these acetylated compounds are illustratedand referred to as compounds #1C to #86C or DC-0001C to DC-0086C; but itshould be understood that the depiction of acetyl esters in Example 31is merely illustrative, and all pharmaceutically acceptable esters areincluded within this invention. The ester groups are expected to serveas intermediate protecting groups for the hydroxyl moieties andtherefore the pharmaceutically acceptable esters are expected to serveas effective prodrugs for their underlying bis- and tris-dihydroxyarylcompounds.

Chemical structures for each of the compounds of this invention (withthe note that the acetates are shown as representative of thepharmaceutically acceptable esters as a class) are shown. The names ofthe compounds are variously IUPAC names [names derived according to theaccepted IUPAC (International Union of Pure and Applied Chemistry)system established by the coalition of the Commission on Nomenclature ofOrganic Chemistry and the Commission on Physical Organic Chemistry; ascan be found at http://www.chem.qmul.ac.uk/iupac, names derived fromIUPAC names by addition or substitution (for example, by the use of“3,4-methylenedioxyphenyl” derived from “phenyl” instead of“benzo[1,3]dioxol-5-yl”), and names derived from the names of reactants(for example, by the use of “3,4-dihydroxybenzoic acid3,4-dihydroxyanilide” instead of“N-(3,4-dihydroxyphenyl)-3,4-dihydroxybenzamide”). However, the namesused are explicitly equated to chemical structures, and are believed tobe readily understood by a person of ordinary skill in the art.

“Mammal” includes both humans and non-human mammals, such as companionanimals (cats, dogs, and the like), laboratory animals (such as mice,rats, guinea pigs, and the like) and farm animals (cattle, horses,sheep, goats, swine, and the like).

“Pharmaceutically acceptable excipient” means an excipient that isconventionally useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use as well as for human pharmaceuticaluse. Such excipients may be solid, liquid, semisolid, or, in the case ofan aerosol composition, gaseous.

“Pharmaceutically acceptable salt” means a salt that is pharmaceuticallyacceptable and have the desired pharmacological properties. Such saltsinclude salts that may be formed where acidic protons present in thecompounds are capable of reacting with inorganic or organic bases.Suitable inorganic salts include those formed with the alkali metals,e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitableorganic salts include those formed with organic bases such as the aminebases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Such salts also include acid additionsalts formed with inorganic acids (e.g. hydrochloric and hydrobromicacids) and organic acids (e.g. acetic acid, citric acid, maleic acid,and the alkane- and arene-sulfonic acids such as methanesulfonic acidand benzenesulfonic acid). When there are two acidic groups present, apharmaceutically acceptable salt may be a mono-acid-mono-salt or adi-salt; and similarly where there are more than two acidic groupspresent, some or all of such groups can be salified.

A “therapeutically effective amount” in general means the amount that,when administered to a subject or animal for treating a disease, issufficient to affect the desired degree of treatment for the disease. A“therapeutically effective amount” or a “therapeutically effectivedosage” preferably inhibits, reduces, disrupts, disassembles amyloid orsynuclein fibril formation, deposition, accumulation and/or persistence,or treats a disease associated with these conditions, such as an amyloiddisease or a synucleinopathy, by at least 20%, more preferably by atleast 40%, even more preferably by at least 60%, and still morepreferably by at least 80%, relative to an untreated subject. Effectiveamounts of a compound of this invention or composition thereof fortreatment of a mammalian subject are about 0.1 to about 1000 mg/Kg ofbody weight of the subject/day, such as from about 1 to about 100mg/Kg/clay, especially from about 10 to about 100 mg/Kg/day. A broadrange of disclosed composition dosages are believed to be both safe andeffective.

“Treating” or “treatment” of a disease includes preventing the diseasefrom occurring in a mammal that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), providing relief from the symptoms or side-effects of thedisease (including palliative treatment), and relieving the disease(causing regression of the disease), such as by disruption of pre-formedamyloid or synuclein fibrils. One such preventive treatment may be useof the disclosed compounds for the treatment of Mild Cognitiveimpairment (MCI).

“NAC” (non-Aβ component) is a 35-amino acid peptide fragment ofα-synuclein, which like α-synuclein, has the ability to formamyloid-like fibrils when incubated at 37° C., and is positive withamyloid stains such as Congo red (demonstrating a red/greenbirefringence when viewed under polarized light) and Thioflavin S(demonstrating positive fluorescence) (Hashimoto et al., Brain Res.799:301-306, 1998; Ueda et al., Proc. Natl. Acad Sci. U.S.A.90:11282-11286, 1993). Inhibition of NAC fibril formation, deposition,accumulation, aggregation, and/or persistence is believed to beeffective treatment for a number of diseases involving α-synuclein, suchas Parkinson's disease, Lewy body disease and multiple system atrophy.

“Fibrillogenesis” refers to the formation, deposition, accumulationand/or persistence of amyloid fibrils, filaments, inclusions, deposits,as well as synuclein (usually involving α-synuclein) and/or NAG fibrils,filaments, inclusions, deposits or the like.

“Inhibition of fibrillogenesis” refers to the inhibition of formation,deposition, accumulation and/or persistence of such amyloid fibrils orsynuclein fibril-like deposits.

“Disruption of fibrils or fibrillogenesis” refers to the disruption ofpre-formed amyloid or synuclein fibrils that usually exist in apre-dominant β-pleated sheet secondary structure. Such disruption bycompounds of the invention may involve marked reduction or disassemblyof amyloid or synuclein fibrils as assessed by various methods such ascircular dichroism spectroscopy, Thioflavin T fluorometry, Congo redbinding, SDS-PAGE/Western blotting, as demonstrated by the Examplespresented in this application.

“A pharmaceutical agent” or “pharmacological agent” or “pharmaceuticalcomposition” refers to a compound or combination of compounds used fortreatment, preferably in a pure or near pure form. In the specification,pharmaceutical or pharmacological agents include the compounds of thisinvention. The compounds are desirably purified to 80% homogeneity, andpreferably to 90% homogeneity. Compounds and compositions purified to99.9% homogeneity are believed to be advantageous. As a test orconfirmation, a suitable homogeneous compound on HPLC would yield whatthose skilled in the art would identify as a single sharp-peak band.

“Neuroprotection” or “neuroprotective” refers to the ability of acompound to protect, reduce, alleviate, ameliorate, and/or attenuatedamage to nerve cells (neurodegeneration).

Compounds of the Invention

The compounds of this invention are:

-   (1) compounds of the formula:

where:

-   R is a C₁-C₁₀ alkylene group, in which, when the number of carbon    atoms is at least 2, there are optionally 1 or 2 non-adjacent double    bonds; 1 to 3 non-adjacent methylene groups are optionally replaced    by NR′ (where R′ is H, alkyl, or acyl), O, or S; and 1 or 2    methylene groups are optionally replaced by a carbonyl or    hydroxymethylene group; and-   (2) the compounds that are:-   3,4,3′,4′-tetrahydroxybenzoin; 3,4,3′,4′-tetrahydroxydesoxybenzoin;    3,4,3′,4′-tetrahydroxydiphenylmethane;    1,2-bis(3,4-dihydroxyphenyl)ethane;    1,3-bis(3,4-dihydroxyphenyl)propane; 3,4,3′,4′-tetrahydroxychalcone;    3,5-bis (3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline;    4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;    1,4-bis(3,4-dihydroxybenzyl)piperazine;    N,N′-bis(3,4-dilrydroxybenzyl)-N,N′-dimethylethylenediamine;    2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane;    N-(3,4-dihydroxybenzyl)proline 3,4-dihydroxybenzylamide;    2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid    3,4-dihydroxy-phenethylamide;    2,6-bis(3,4-dihydroxybenzyl)cyclohexanone;    3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone;    2,4-bis(3,4-dihydroxybenzyl)-3-tropinone;    tris(3,4-dihydroxybenzyl)methane;    α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid    3,4-dihydroxybenzyl amide;    4-(3,4-dihydroxy-benzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one;    1,4-bis(3,4-dihydroxybenzoyl)piperazine;    N,N′-bis(3,4-dihydroxybenzoyl)-N,N′-dimethylethylenediamine;    2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane;    N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane;    3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine;    3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine;    N-(3,4-dihydroxyphenylacetyl)proline-3,4-dihydroxyanilide;    2,3-bis(3,4-dihydroxyphenyl)butane;    1,3-bis(3,4-dihydroxybenzyl)benzene;    1,4-bis(3,4-dihydroxybenzyl)benzene;    2,6-bis(3,4-dihydroxybenzyl)-pyridine;    2,5-bis(3,4-dihydroxybenzyl)thiophene;    2,3-bis(3,4-dihydroxybenzyl)thiophene;    1,2-bis(3,4-dihydroxyphenyl)cyclohexane;    1,4-bis(3,4-dihydroxyphenyl)cyclohexane;    3,7-bis(3,4-dihydroxy-phenyl)bicyclo[3.3.0]octane;    2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethyl-bicyclo[2.2.1]heptane;    1,2-bis(3,4-dihydroxyphenoxy)ethane;    1,3-bis(3,4-dihydroxyphenoxy)propane;    trans-1,2-bis(3,4-dihydroxy-phenoxy)cyclopentane;    N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxy-benzylamide;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyphenethylamide;    3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide;    3,4-dihydroxybenzoic acid o-(3,4-dihydroxyphenoxy)anilide;    2,6-bis(3,4-dihydroxyphenoxy)pyridine; 3,4-dihydroxybenzoic acid    3,4-dihydroxyanilide; 3,4-dihydroxybenzoic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid    3,4-dihydroxyphenethylamide; 3,4-dihydroxyphenyl acetic acid    3,4-dihydroxyanilide; 3,4-dihydroxyphenylacetic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxyphenylacetic acid    3,4-dihydroxyphenethylamide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxyphenethylamide; 3,4-dihydroxycinnamic acid    3,4-dihydroxyanilide; 3,4-dihydroxycinnamic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxycinnamic acid    3,4-dihydroxyphenethylamide; oxalic acid bis(3,4-dihydroxyanilide);    oxalic acid bis(3,4-dihydroxybenzylamide); oxalic acid    bis(3,4-dihydroxyphenethylamide); succinic acid    bis(3,4-dihydroxyanilide); succinic acid    bis(3,4-dihydroxybenzylamide); succinic acid    bis(3,4-dihydroxyphenethylamide); maleic acid    bis(3,4-dihydroxyanilide); maleic acid bis(3,4dihydroxybenzylamide);    fumaric acid bis(3,4-dihydroxyanilide); furnaric acid    bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine;    N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine;    tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea;    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea;    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;    3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin;    3-deoxy-3-(3,4dihydroxyphenethyl)aminoepicatechin;    2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine;    10-aminoanthracene-1,2,7,8-tetraol; acridine-1,2,6,7-tetraol;    phenoxazine-2,3,7,8,10-pentaol;    dibenzo[c,f][2,7]napthyridine-2,3,10,11-tetraol; and    6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-2,10,11-triol;-   (3) the methylenedioxy analogs and pharmaceutically acceptable    esters of the compounds of (1) and (2); and-   (4) the pharmaceutically acceptable salts of the compounds of (1) to    (3).

Within the compounds of this invention, a first group of compounds isthe compounds selected from the group consisting of:

-   (1) compounds of the formula:

where:

-   R is a C₁-C₁₀, especially a C₁₋₆, alkylene group, in which, when the    number of carbon atoms is at least 2, there are optionally 1 or 2    non-adjacent double bonds; 1 to 3 non-adjacent methylene groups are    optionally replaced by NR′ (where R′ is H, C₁₋₃ alkyl, or C₂₋₄    acyl), O, or S, especially NH or N—CH₃; and 1 or 2 methylene groups    are optionally replaced by a carbonyl or hydroxymethylene group;-   (2) the methylenedioxy analogs and pharmaceutically acceptable    tetraesters thereof; and-   (3) the pharmaceutically acceptable salts of the compounds of (1)    and (2).

Within this first group, a subgroup of compounds is the group ofcompounds selected from the group consisting of:

-   (1) compounds of the formula:

where:

-   R is a C₂-C₁₀, especially a C₂₋₆, alkylene group, in which there is    optionally 1 double bond; and 1 or 2 non-adjacent ethylene groups    are replaced by —C(O)NR′— or —NR′C(O)— (where R′ is H or lower    alkyl);-   (2) the methylenedioxy analogs and pharmaceutically acceptable    tetraesters thereof; and-   (3) the pharmaceutically acceptable sans of compounds of (1) and    (2).

Within the compounds of this invention, a second group of compounds is:

-   (1) the compounds that are:-   3,4,3′,4′-tetrahydroxybenzoin; 3,4,3′,4′-tetrahydroxydesoxybenzoin;    3,4,3′,4′-tetrahydroxydiphenylmethane;    1,2-bis(3,4-dihydroxyphenyl)ethane;    1,3-bis(3,4-dihydroxyphenyl)propane; 3,4,3′,4′-tetrahydroxychalcone;    3,5-bis(3,4-dihydroxyphenyl)-1- methyl-2-pyrazoline;    4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;    1,4-bis(3,4-dihydroxybenzyl)piperazine;    N,N′-bis(3,4-dihydroxybenzyl)-N,N′-dimethylethylenediamine;    2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane;    N-(3,4-dihydroxybenzyl)proline 3,4-dihydroxybenzylamide;    2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid    3,4-dihydroxy-phenethylamide;    2,6-bis(3,4-dihydroxybenzyl)cyclohexanone;    3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone;    2,4-bis(3,4-dihydroxybenzyl)-3-tropinone;    tris(3,4-dihydroxybenzyl)methane;    α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid    3,4-dihydroxybenzyl amide;    4-(3,4-dihydroxy-benzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one;    1,4-bis(3,4-dihydroxybenzoyl)piperazine;    N,N′-bis(3,4-dihydroxybenzoyl)-N,N′-dimethylethylenediamine;    2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane;    N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane;    3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine;    3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine;    N-(3,4-dihydroxyphenylacetyl)proline-3,4-dihydroxyanilide;    2,3-bis(3,4-dihydroxyphenyl)butane;    1,3-bis(3,4-dihydroxybenzyl)benzene;    1,4-bis(3,4-dihydroxybenzyl)benzene;    2,6-bis(3,4-dihydroxybenzyl)-pyridine;    2,5-bis(3,4-dihydroxybenzyl)thiophene;    2,3-bis(3,4-dihydroxybenzyl)thiophene;    1,2-bis(3,4-dihydroxyphenyl)cyclohexane;    1,4-bis(3,4-dihydroxyphenyl)cyclohexane;    3,7-bis(3,4-dihydroxy-phenyl)bicyclo[3.3.0]octane;    2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethyl-bicyclo[2.2.1]heptane;    1,2-bis(3,4-dihydroxyphenoxy)ethane;    1,3-bis(3,4-dihydroxyphenoxy)propane;    trans-1,2-bis(3,4-dihydroxy-phenoxy)cyclopentane;    N-(3,4-dihydroxybenzy-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxy-benzylamide;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyphenethylamide;    3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide;    3,4-dihydroxybenzoic acid o-(3,4-dihydroxyphenoxy)anilide;    2,6-bis(3,4-dihydroxyphenoxy)pyridine; 3,4-dihydroxybenzoic acid    3,4-dihydroxyanilide; 3,4-dihydroxybenzoic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid    3,4-dihydroxyphenethylamide; 3,4-dihydroxyphenyl acetic acid    3,4-dihydroxyanilide; 3,4-dihydroxyphenylacetic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxyphenylacetic acid    3,4-dihydroxyphenethylamide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxyphenethylamide; 3,4-dihydroxycinnamic acid    3,4-dihydroxyanilide; 3,4-dihydroxycinnamic acid    3,4dihydroxybenzylamide; 3,4-dihydroxycinnamic acid    3,4-dihydroxyphenethylamide; oxalic acid bis(3,4-dihydroxyanilide);    oxalic acid bis(3,4-dihydroxybenzylamide); oxalic acid    bis(3,4-dihydroxyphenethylamide); succinic acid    bis(3,4-dihydroxyanilide); succinic acid    bis(3,4-dihydroxybenzylamide); succinic acid    bis(3,4-dihydroxyphenethylamide); maleic acid    bis(3,4-dihydroxyanilide); maleic acid    bis(3,4-dihydroxybenzylamide); fumaric acid    bis(3,4-dihydroxyanilide); fumaric acid    bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine;    N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine;    tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea;    (3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea;    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;    3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin;    3-deoxy-3-(3,4-dihydroxyphenethyl)aminoepicatechin;    2,3,6,7-tetrahydroxy-9,10-epoxy-9,10-dihydroacridine;    10-aminoanthracene-1,2,7,8-tetraol; acridine-1,2,6,7-tetraol;    phenoxazine-2,3,7,8,10-pentaol;    dibenzo[c,f][2,7]napthyridine-2,3,10,11-tetraol; and    6-methyl-5,6,6a,7-tetrahydro-4H-1-dibenzo[de,g]quinoline-2,10,11-triol;-   (2) the methylenedioxy analogs and pharmaceutically acceptable    esters thereof; and-   (3) the pharmaceutically acceptable salts of the compounds of (1)    and (2).

Within this second group, a subgroup of compounds is:

-   (1) the compounds that are:-   3,4,3′,4′-tetrahydroxybenzoin; 3,4,3′,4′-tetrahydroxydesoxybenzoin;    3,4,3′,4″-tetrahydroxydiphenylmethane;    1,2-bis(3,4-dihydroxyphenyl)ethane;    1,3-bis(3,4-dihydroxyphenyl)propane; 3,4,3′,4′-tetrahydroxychalcone;    3,5-bis(3,4-dihydroxyphenyl)-1-methyl-2-pyrazoline;    4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;    1,4-bis(3,4-dihydroxybenzyl)piperazine;    N,N′-bis(3,4-dihydroxybenzyl)-N,N′-dimethylethylenediamine;    2,5-bis(3,4-dihydroxybenzyl)-2,5-diaza[2.2.1]bicycloheptane;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,4-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzyl)-cis-1,3-bis(aminomethyl)cyclohexane;    N-(3,4-dihydroxybenzyl)proline 3,4-dihydroxybenzylamide;    2-(3,4-dihydroxybenzyl)isoquinoline-3-carboxylic acid    3,4-dihydroxy-phenethylamide;    2,6-bis(3,4-dihydroxybenzyl)cyclohexanone;    3,5-bis(3,4-dihydroxybenzyl)-1-methyl-4-piperidinone;    2,4-bis(3,4-dihydroxybenzyl)-3-tropinone;    tris(3,4-dihydroxybenzyl)methane;    α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid    3,4-dihydroxybenzyl amide;    4-(3,4-dihydroxy-benzylaminomethylene)-2-(3,4-dihydroxyphenyl)oxazolin-5-one;    1,4-bis(3,4-dihydroxybenzoyl)piperazine;    N,N′-bis(3,4-dihydroxybenzoyl)-N,N′-dimethylethylenediamine;    2,5-bis(3,4-dihydroxybenzoyl)-2,5-diaza[2.2.1]bicycloheptane;    N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane;    N,N′-bis(3,4-dihydroxybenzoyl)-cis-1,3-bis(aminomethyl)cyclohexane;    3,6-bis(3,4-dihydroxybenzyl)-2,5-diketopiperazine;    3,6-bis(3,4-dihydroxybenzylidene)-1,4-dimethyl-2,5-diketopiperazine;    N-(3,4-dihydroxyphenylacetyl)proline-3,4-dihydroxyanilide;    2,3-bis(3,4-dihydroxyphenyl)butane;    1,3-bis(3,4-dihydroxybenzyl)benzene;    1,4-bis(3,4-dihydroxybenzyl)benzene;    2,6-bis(3,4-dihydroxybenzyl)-pyridine;    2,5-bis(3,4-dihydroxybenzyl)thiophene;    2,3-bis(3,4-dihydroxybenzyl)thiophene;    1,2-bis(3,4-dihydroxyphenyl)cyclohexane;    1,4-bis(3,4-dihydroxyphenyl)cyclohexane;    3,7-bis(3,4-dihydroxy-phenyl)bicyclo[3.3.0]octane;    2,3-bis(3,4-dihydroxyphenyl)-1,7,7-trimethyl-bicyclo[2.2.1]heptane;    1,2-bis(3,4-dihydroxyphenoxy)ethane;    1,3-bis(3,4-dihydroxyphenoxy)propane;    trans-1,2-bis(3,4-dihydroxy-phenoxy)cyclopentane;    N-(3,4-dihydroxybenzyl)-3-(3,4-dihydroxyphenoxy)-2-hydroxypropylamine;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyanilide;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxy-benzylamide;    3,4-dihydroxyphenoxyacetic acid 3,4-dihydroxyphenethylamide;    3,4-dihydroxybenzoic acid p-(3,4-dihydroxyphenoxy)anilide;    3,4-dichloroxybenzoic acid o-(3,4-dihydroxyphenoxy)anilide;    2,6-bis(3,4-dihydroxyphenoxy)pyridine; 3,4-dihydroxybenzoic acid    3,4-dihydroxyanilide; 3,4-dihydroxybenzoic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid    3,4-dihydroxyphenethylamide; 3,4-dihydroxyphenyl acetic acid    3,4-dihydroxyanilide; 3,4-dihydroxyphenylacetic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxyphenylacetic acid    3,4-dihydroxyphenethylamide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxyphenethylamide; 3,4-dihydroxycinnamic acid    3,4-dihydroxyanilide; 3,4-dihydroxycinnamic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxycinnamic acid    3,4-dihydroxyphenethylamide; oxalic acid bis(3,4-dihydroxyanilide);    oxalic acid bis(3,4-dihydroxybenzylamide); oxalic acid    bis(3,4-dihydroxyphenethylamide); succinic acid    bis(3,4-dihydroxyanilide); succinic acid    bis(3,4-dihydroxybenzylamide); succinic acid    bis(3,4-dihydroxyphenethylamide); maleic acid    bis(3,4-dihydroxyanilide); maleic acid    bis(3,4-dihydroxybenzylamide); fumaric acid    bis(3,4-dihydroxyanilide); fumaric acid    bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine;    N-(3,4-dihydroxybenzyl)-3,4-dihydroxyphenethylamine;    tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea;    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea;    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;    3-deoxy-3-(3,4-dihydroxybenzyl)aminoepicatechin; and    3-deoxy-3-(3,4-dihydroxyphenethyl)aminoepicatechin;-   (2) the methylenedioxy analogs and pharmaceutically acceptable    esters thereof; and-   (3) the pharmaceutically acceptable salts of the compounds of (1)    and (2).

Within this subgroup, a further subgroup is:

-   (1) the compounds that are:-   3,4,3′,4′-tetrahydroxybenzoin;    3,4,3′,4′-tetrahydroxydiphenylmethane;    1,2-bis(3,4-dihydroxyphenyl)ethane;    4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine;    1,4-bis(3,4-dihydroxybenzyl)piperazine;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane;    2,4-bis(3,4-dihydroxybenzyl)-3-tropinone;    α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid    3,4-dihydroxybenzyl amide; 1,4-bis(3,4-dihydroxybenzoyl)piperazine;    N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane;    3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide; 3,4-dihydroxybenzoic    acid 3,4-dihydroxybenzylamide; dihydroxyphenyl)propionic acid    3,4-dihydroxyanilide; 3-(3,4-dihydroxyphenyl)propionic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxycinnamic acid    3,4-dihydroxybenzylamide; oxalic acid bis(3,4-dihydroxyanilide);    succinic acid bis(3,4-dihydroxyanilide); succinic acid    bis(3,4-dihydroxybenzylamide); bis(3,4-dihydroxybenzyl)amine;    tris(3,4-dihydroxybenzyl)amine; 1,3-bis(3,4-dihydroxyphenyl)urea;    and 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;-   (2) the methylenedioxy analogs and pharmaceutically acceptable    esters thereof; and-   (3) the pharmaceutically acceptable salts of the compounds of (1)    and (2).

Within each of these groups and subgroups, there are especially thecompounds of the invention that are the bis- and tris(dihydroxyaryl)compounds (i.e. the compounds of the formula or of the list) andcompound #86, and their pharmaceutically acceptable salts.

Synthesis of the Compounds of the Invention

The compounds of this invention may be prepared by methods generallyknown to the person of ordinary skill in the art, having regard to thatknowledge and the disclosure of this application including Examples1-24.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as the AldrichChemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma(St. Louis, Mo.), or Lancaster Synthesis Inc. (Windham, N.H.) or areprepared by methods well known to a person of ordinary skill in the art,following procedures described in such references as Fieser and Fieser'sRegents for Organic Synthesis, vols. 1-17, John Wiley and Sons, NewYork, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 andsupps., Elsevier Science Publishers, 1989; Organic Reactions, vols.1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: AdvancedOrganic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; andLarock: Comprehensive Organic Transformations, VCH Publishers, New York,1989.

In most cases, protective groups for the hydroxy groups are introducedand finally removed. Suitable protective groups are described in Greeneet al., Protective Groups in Organic Synthesis, Second Edition, JohnWiley and Sons, New York, 1991. A preferred protective group is themethylenedioxy group, as seen in many of Examples 1-23, and a widevariety of methylenedioxyphenyl compounds (such as3,4-methylenedioxyacetophenone, 3,4-methylenedioxyaniline,3,4-methylenedioxybenzaldehyde, 3,4-methylenedioxybenzoic acid,3,4-methylenedioxybenzonitrile, 3,4-methylenedioxybenzoic acid,3,4-methylenedioxybenzoyl chloride, 3,4-methylenedioxycinnamic acid,3,4-methylenedioxynitrobenzene, 3,4-methylenedioxyphenol,3,4-methylenedioxyphenylacetic acid,3,4-methylenedioxyphenylacetonitrile, 3,4-methylenedioxyphenylisocyanate, 3,4-methylenedioxyphenylmagnesium bromide, and3,4-methylenedioxyphenylmethanol) are commercially available. Otherprotecting groups, such as the benzyl and methoxymethyl groups, may alsobe used

Other starting materials or early intermediates may be prepared byelaboration of the materials listed above, for example, by methods wellknown to a person of ordinary skill in the art.

The starting materials, intermediates, and compounds of this inventionmay be isolated and purified using conventional techniques, includingprecipitation, filtration, distillation, crystallization,chromatography, and the like. The compounds may be characterized usingconventional methods, including physical constants and spectroscopicmethods.

Pharmacology and Utility

The compounds of this invention, either as the dihydroxyaryl compoundsper se, or as the methylenedioxy analogs or pharmaceutically acceptableesters (once de-protected either in the body or in vitro), act toinhibit or prevent amyloid fibril formation, inhibit or prevent amyloidfibril growth, and/or cause disassembly, disruption, and/ordisaggregation of pre-formed amyloid fibrils and amyloid proteindeposits including α-synuclein. Their activity can be measured in tiroby methods such as those discussed in Examples 25-27, and 35 while theiractivity in vivo against amyloid diseases can be measured in animalmodels, such as those APP transgenic mouse models that mimic many of theneuropathological hallmarks of Alzheimer's disease, and in humans. Thesame in vitro methods can be used to detect the activity of thecompounds of this invention on NAC fibril formation, deposition,accumulation, aggregation, and/or persistence. Examples 33 and 34summarize in vivo methods whereby α-synuclein fibril formation,deposition, accumulation, aggregation, and/or persistence can bemeasured in animal or cellular models that mimic many of theneuropathological characteristics of diseases involving α-synuclein,such as Parkinson's disease, Lewy body disease and multiple systematrophy.

“Amyloid diseases” or “amyloidoses” suitable for treatment with thecompounds of this invention are diseases associated with the formation,deposition, accumulation, or persistence of amyloid fibrils, especiallythe fibril' s of an amyloid protein selected from the group consistingof Aβ amyloid, AA amyloid, AL amyloid, IAPP amyloid, PrP amyloid,α₂-microglobulin amyloid, transthyretin, prealbumin, and procalcitonin,especially Aβ amyloid and IAPP amyloid. Suitable such diseases includeAlzheimer's disease, Down's syndrome, dementia pugilistica, multiplesystem atrophy, inclusion body myositosis, hereditary cerebralhemorrhage with amyloidosis of the Dutch type, Nieman-Pick disease typeC, cerebral β-amyloid angiopathy, dementia associated with corticalbasal degeneration, the amyloidosis of type 2 diabetes, the amyloidosisof chronic inflammation, the amyloidosis of malignancy and FamilialMediterranean Fever, the amyloidosis of multiple myeloma and B-celldyscrasias, the amyloidosis of the prion diseases, Creutzfeldt-Jakobdisease, Gerstmann-Straussler syndrome, kuru, scrapie, the amyloidosisassociated with carpal tunnel syndrome, senile cardiac amyloidosis,familial amyloidotic polyneuropathy, and the amyloidosis associated withendocrine tumors, especially Alzheimer's disease and type 2 diabetes.

The compounds also act to inhibit or prevent α-synuclein/NAC fibrilformation, inhibit or prevent α-synuclein/NAC fibril growth, and/orcause disassembly, disruption, and/or disaggregation of preformedα-synuclein/NAC fibrils and α-synuclein/NAC-associated protein deposits.Their activity can be measured in vitro by methods similar to thosediscussed in Examples 24-26, or in vivo in animal models, such as thoseα-synuclein transgenic mouse models that mimic some of theneuropathological hallmarks of Parkinson's disease or in cell culturesystems.

“Synuclein diseases” or “synucleinopathies” suitable for treatment withthe compounds of this invention are diseases associated with theformation, deposition, accumulation, or persistence of synucleinfibrils, especially α-synuclein fibrils. Suitable such diseases includeParkinson's disease, familial Parkinson's disease, Lewy body disease,the Lewy body variant of Alzheimer's disease, dementia with Lewy bodies,multiple system atrophy, and the Parkinsonism-dementia complex of Guam.

The therapeutic ratio of a compound can be determined, for example, bycomparing the dose that gives effective anti-fibril (anti-amyloid oranti-α-synuclein/NAC) activity in a suitable in sivo model in a suitableanimal species such as the mouse, with the dose that gives significantweight loss (or other observable side-effects) in the test animalspecies.

Compounds of special interest for treating the formation, deposition,accumulation, or persistence of Aβ amyloid fibrils, or α-synucleinfibrils for treating Alzheimer's disease or Parkinson's disease, areselected from the group consisting of (1) the compounds that are:

-   3,4,3′,4′-tetrahydroxybenzoin;    3,4,3′,4′-tetrahydroxydiphenylmethane;    1,2-bis(3,4-dihydroxyphenyl)ethane;    N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane;    α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid    3,4-dihydroxyanilide; bis(3,4-dihydroxybenzy)amine;    1,3-bis(3,4-dihydroxyphenyl)urea; and    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;    1,4-bis(3,4-dihydroxybenzoyl)piperazine; oxalic acid    bis(3,4-dihydroxyanilide)-   (2) the methylenedioxy analogs and pharmaceutically acceptable    esters thereof; and-   (3) the pharmaceutically acceptable salts of the compounds of (1)    and (2).    Especially of interest are the compounds of (1) above and their    pharmaceutically acceptable salts.

Compounds of special interest for treating the formation, deposition,accumulation, or persistence of IAPP amyloid fibrils, or for treatingtype 2 diabetes, are selected from the group consisting of (1) thecompounds that are: 3,4,3′,4′-tetrahydroxybenzoin;3,4,3′,4′-tetrahydroxydiphenylmethane;1,2-bis(3,4-dihydroxyphenyl)ethane;2,4-bis(3,4-dihydroxybenzyl)-3-tropinone; 1,4-bis(3,4-dihydroxybenzoyl)piperazine; 3,4-dihydroxybenzoic acid3,4-dihydroxyanilide; 3,4-dihydroxybenzoic acid3,4-dihydroxybenzylamide; 3-(3,4-dihydroxyphenyl)propionic acid3,4-dihydroxybenzylamide; oxalic acid bis(3,4-dihydroxyanilide);succinic acid bis(3,4-dihydroxyanilide); tris(3,4-dihydroxybenzyl)amine;and 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;

-   (2) the methylenedioxy analogs and pharmaceutically acceptable    esters thereof; and-   (3) the pharmaceutically acceptable salts of the compounds of (1)    and (2).

Especially of interest are the compounds of (1) above and theirpharmaceutically acceptable salts.

Compounds of special interest for treating the formation, deposition,accumulation, or persistence of α-synuclein fibrils, or for treatingParkinson's disease or other synuckinopathies, are selected from thegroup consisting of (1) the compounds that are:

-   3,4,3′,4′-tetrahydroxybenzoin;    3,4,3′,4′-tetrahydroxydiphenylmethane; 1,2-    bis(3,4-dihydroxyphenyl)ethane; N,N′-bis(3,4-    dihydroxybenzyl)-trans-1,2-diaminocyclohexane;    α-(3,4-dihydroxybenzamido)-3,4-dihydroxycinnamic acid    3,4-dihydroxybenzylamide; 3,4-dihydroxybenzoic acid    3,4-dihydroxyanilide; bis(3,4-dihydroxybenzyl)amine;    1,3-bis(3,4-dihydroxyphenyl)urea; and    1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea;    1,4-bis(3,4-dihydroxybenzoyl)piperazine; oxalic acid    bis(3,4-dihydroxyanilide)-   (2) the methylenedioxy analogs and pharmaceutically acceptable    esters thereof; and-   (3) the pharmaceutically acceptable salts of the compounds of (1)    and (2).

Especially of interest are the compounds of (1) above and theirpharmaceutically acceptable salts.

Pharmaceutical Compositions and Administration

In general, compounds of the invention will be administered intherapeutically effective amounts by any of the usual modes known in theart, either singly or in combination with at least one other compound ofthis invention and/or at least one other conventional therapeutic agentfor the disease being treated. A therapeutically effective amount mayvary widely depending on the disease, its severity, the age and relativehealth of the animal being treated, the potency of the compound(s), andother factors. As anti-fibril agents, therapeutically effective amountsof compounds of this invention may range from 0.1-1000 mg/Kg bodyweight/day, such as from 1-100 mg/Kg/day; for example, 10-100 mg/Kg/day.A person of ordinary skill in the art will be conventionally able, andwithout undue experimentation, having regard to that skill and to thisdisclosure, to determine a therapeutically effective amount of acompound for the treatment of an amyloid disease such as an amyloidosisor α-synuclein/NAC fibril formation.

Preferred compositions will contain a compound of this invention that isat least substantially pure. In general “pure” means better than 95%pure, and “substantially pure” means a compound synthesized such thatthe compound, as made as available for consideration into a therapeuticdosage, has only those impurities that can not readily nor reasonably beremoved by conventional purification processes.

In general, the compounds of this invention will be administered aspharmaceutical compositions by one of the following routes: oral,topical, systemic (e.g. transdermal, intranasal, or by suppository), orparenteral (e.g. intramuscular, subcutaneous, or intravenous injection).Compositions may take the form of tablets, pills, capsules, semisolids,powders, sustained release formulations, solutions, suspensions,elixirs, aerosols, or any other appropriate compositions; and compriseat least one compound of this invention in combination with at least onepharmaceutically acceptable excipient. Suitable excipients are wellknown to persons of ordinary skill in the art, and they, and the methodsof formulating the compositions, may be found in such standardreferences as Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins,Philadelphia, Pa. Suitable liquid carriers, especially for injectablesolutions, include water, aqueous saline solution, aqueous dextrosesolution, and glycols.

In particular, the compound(s)—optimally only one such compound isadministered in any particular dosage form—can be administered, orally,for example, as tablets, troches, lozenges, aqueous or oily suspension,dispersible powders or granules, emulsions, hard or soft capsules, orsyrups or elixirs. Compositions intended for oral use may be preparedaccording to any method known in the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations.

Tablets contain the compound in admixture with non-toxicpharmaceutically acceptable excipients that are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, maize starch or alginic acid; binding agents, for example,maize starch, gelatin or acacia, and lubricating agents, for example,magnesium stearate or stearic acid or tale. The tablets may be uncoatedor they may be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglycerol monostearate or glycerol distearate may be employed.Formulations for oral use may also be presented as hard gelatin capsuleswherein the compound is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin., or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the compound in admixture with excipientssuitable for the manufacture of aqueous suspensions. Such excipients aresuspending agents, for example, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethyl cellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents may be naturally occurring phosphatides, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for example,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids such as hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters from fatty acids and a hexitolanhydrides, for example, polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for example,ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, or one or more sweetening agents, such as sucroseor saccharin.

Oily suspensions may be formulated by suspending the compound in avegetable oil, for example arachis oil, olive oil, sesame oil, orcoconut oil or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents, such as those set forthbelow, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid. Dispersible powders and granulessuitable for preparation of an aqueous suspension by the addition ofwater provide the active ingredient in admixture with a dispersing orwetting agent, a suspending agent and one or more preservatives.Suitable dispersing or wetting agents and suspending agents areexemplified by those already described above. Additional excipients, forexample sweetening, flavoring and agents, may also be present.

The compounds of the invention may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil, for example olive oilor arachis oils, or a mineral oil, for example liquid paraffin ormixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally occurring phosphatides, for example soy bean, lecithin, andoccurring phosphatides, for example soy bean, lecithin, and esters orpartial esters derived from fatty acids and hexitol anhydrides, forexample sorbitan monooleate, and condensation products of the saidpartial esters with ethylene oxide, for example polyoxyethylene sorbitanmonooleate. The emulsion may also contain sweetening and flavoringagents. Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, sorbitol or sucrose. Such formulations may alsocontain a demulcent, a preservative and flavoring and coloring agents.

The compounds of the invention can also be administered by injection orinfusion, either subcutaneously or intravenously, or intramuscularly, orintrasternally, or intranasally, or by infusion techniques in the formof sterile injectable or oleaginous suspension. The compound may be inthe form of a sterile injectable aqueous or oleaginous suspensions.These suspensions may be formulated according to the known art usingsuitable dispersing of wetting agents and suspending agents that havebeen described above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilsmay be conventionally employed including synthetic mono- ordiglyterides. In addition fatty acids such as oleic acid find use in thepreparation of injectables. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided dosagesmay be administered daily or the dosage may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

It is especially advantageous to formulate the compounds in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each containing atherapeutically effective quantity of the compound and at least onepharmaceutical excipient. A drug product will comprise a dosage unitform within a container that is labeled or accompanied by a labelindicating the intended method of treatment, such as the treatment of anamyloid disease, for example an amyloidosis such as Alzheimer's diseaseor a disease associated with α-synuclein/NAC fibril formation such asParkinson's disease.

Sustained Release Formulations

The invention also includes the use of sustained release formulations todeliver the compounds of the present invention to the desired target(i.e. brain or systemic organs) at high circulating levels (between 10⁻⁹and 10⁻⁴M) are also disclosed. In a preferred embodiment for thetreatment of Alzheimer's or Parkinson's disease, the circulating levelsof the compounds is maintained up to 10⁻⁷ M. The levels are eithercirculating in the patient systemically, or in a preferred embodiment,present in brain tissue, and in a most preferred embodiments, localizedto the amyloid or α-synuclein fibril deposits in brain or other tissues.

It is understood that the compound levels are maintained over a certainperiod of time as is desired and can be easily determined by one skilledin the art using this disclosure and compounds of the invention. In apreferred embodiment, the invention includes a unique feature ofadministration comprising a sustained release formulation so that aconstant level of therapeutic compound is maintained between 10⁻⁸ and10⁻⁶ M between 48 to 96 hours in the sera.

Such sustained and/or timed release formulations may be made bysustained release means of delivery devices that are well known to thoseof ordinary skill in the art, such as those described in U.S. Pat. Nos.3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556 and 5,733,566, the disclosures of which are each incorporatedherein by reference. These pharmaceutical compositions can be used toprovide slow or sustained release of one or more of the active compoundsusing, for example, hydroxypropylmethyl cellulose, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or the like. Suitablesustained release formulations known to those skilled in the art,including those described herein, may be readily selected for use withthe pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for oral administration, such as, but not limitedto, tablets, capsules, gelcaps, caplets, powders and the like, that areadapted for sustained release are encompassed by the present invention.

In a preferred embodiment, the sustained release formulation containsactive compound such as, but not limited to, microcrystalline cellulose,maltodextrin, ethylcellulose, and magnesium stearate. As describedabove, all known methods for encapsulation which are compatible withproperties of the disclosed compounds are encompassed by this invention.The sustained release formulation is encapsulated by coating particlesor granules of the pharmaceutical composition of the invention withvarying thickness of slowly soluble polymers or by microencapsulation.In a preferred embodiment, the sustained release formulation isencapsulated with a coating material of varying thickness (e.g. about 1micron to 200 microns) that allow the dissolution of the pharmaceuticalcomposition about 48 hours to about 72 hours after administration to amammal. In another embodiment, the coating material is a food-approvedadditive.

In another embodiment, the sustained release formulation is a matrixdissolution device that is prepared by compressing the drug with aslowly soluble polymer carrier into a tablet. In one preferredembodiment, the coated particles have a size range between about 0.1 toabout 300 microns, as disclosed in U.S. Pat. Nos. 4,710,384 and5,354,556, which are incorporated herein by reference in theirentireties. Each of the particles is in the form of a micromatrix, withthe active ingredient uniformly distributed throughout the polymer.

Sustained release formulations such as those described in U.S. Pat. No.4,710,384, which is incorporated herein by reference in its entirety,having a relatively high percentage of plasticizer in the coating inorder to permit sufficient flexibility to prevent substantial breakageduring compression are disclosed. The specific amount of plasticizervaries depending on the nature of the coating and the particularplasticizer used. The amount may be readily determined empirically bytesting the release characteristics of the tablets formed. If themedicament is released too quickly, then more plasticizer is used.Release characteristics are also a function of the thickness of thecoating. When substantial amounts of plasticizer are used, the sustainedrelease capacity of the coating diminishes. Thus, the thickness of thecoating may be increased slightly to make up for an increase in theamount of plasticizer. Generally, the plasticizer in such an embodimentwill be present in an amount of about 15 to 30% of the sustained releasematerial in the coating, preferably 20 to 25%, and the amount of coatingwill be from 10 to 25% of the weight of the active material. Preferably15 to 20%. Any conventional pharmaceutically acceptable plasticizer maybe incorporated into the coating.

The compounds of the invention can be formulated as a sustained and/ortimed release formulation. All sustained release pharmaceutical productshave a common goal of improving drug therapy over that achieved by theirnon-sustained counterparts. Ideally, the use of an optimally designedsustained release preparation in medical treatment is characterized by aminimum of drug substance being employed to cure or control thecondition. Advantages of sustained release formulations may include: 1)extended activity of the composition, 2) reduced dosage frequency, and3) increased patient compliance. In addition, sustained releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the composition, and thus canaffect the occurrence of side effects.

The sustained release formulations of the invention are designed toinitially release an amount of the therapeutic composition that promptlyproduces the desired therapeutic effect, and gradually and continuallyrelease of other amounts of compositions to maintain this level oftherapeutic effect over an extended period of time. In order to maintainthis constant level in the body, the therapeutic composition must bereleased from the dosage form at a rate that will replace thecomposition being metabolized and excreted from the body.

The sustained release of an active ingredient may be stimulated byvarious inducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “sustained releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, microspheres, orthe like, or a combination thereof, that facilitates the sustainedrelease of the active ingredient.

If the complex is water-soluble, it may be formulated in an appropriatebuffer, for example, phosphate buffered saline, or other physiologicallycompatible solutions. Alternatively, if the resulting complex has poorsolubility in aqueous solvents, then it may be formulated with anon-ionic surfactant such as Tween, or polyethylene glycol. Thus, thecompounds and their physiologically solvents may be formulated foradministration by inhalation or insufflation (either through the mouthor the nose) or oral, buccal, parenteral, or rectal administration, asexamples.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. In a preferred embodiment,the compounds of the present invention are formulated as controlledrelease powders of discrete microparticles that can be readilyformulated in liquid form. The sustained release powder comprisesparticles containing an active ingredient and optionally, an excipientwith at least one non-toxic polymer.

The powder can be dispersed or suspended in a liquid vehicle and willmaintain its sustained release characteristics for a useful period oftime. These dispersions or suspensions have both chemical stability andstability in terms of dissolution rate. The powder may contain anexcipient comprising a polymer, which may be soluble, insoluble,permeable, impermeable, or biodegradable. The polymers may be polymersor copolymers. The polymer may be a natural or synthetic polymer.Natural polymers include polypeptides (e.g., zein), polysaccharides(e.g., cellulose), and alginic acid. Representative synthetic polymersinclude those described, but not limited to, those described in column3, lines 33-45 of U.S. Pat. No. 5,354,556, which is incorporated byreference in its entirety. Particularly suitable polymers include thosedescribed, but not limited to those described in column 3, line46-column 4, line 8 of U.S. Pat. No. 5,354,556 which is incorporated byreference in its entirety.

The sustained release compounds of the invention may be formulated forparenteral administration, e.g., by intramuscular injections or implantsfor subcutaneous tissues and various body cavities and transdermaldevices. In one embodiment, intramuscular injections are formulated asaqueous or oil suspensions. In an aqueous suspension, the sustainedrelease effect is due to, in part, a reduction in solubility of theactive compound upon complexation or a decrease in dissolution rate. Asimilar approach is taken with oil suspensions and solutions, whereinthe release rate of an active compound is determined by partitioning ofthe active compound out of the oil into the surrounding aqueous medium.Only active compounds which are oil soluble and have the desiredpartition characteristics are suitable. Oils that may be used forintramuscular injection include, but are not limited to, sesame, olive,arachis, maize, almond, soybean, cottonseed and castor oil.

A highly developed form of drug delivery that imparts sustained releaseover periods of time ranging from days to years is to implant adrug-bearing polymeric device subcutaneously or in various bodycavities. The polymer material used in an implant, which must bebiocompatible and nontoxic, include but are not limited to hydrogels,silicones, polyethylenes, ethylene-vinyl acetate copolymers, orbiodegradable polymers.

The following non-limiting Examples are given by way of illustrationonly and are not considered a limitation of this invention, manyapparent variations of which are possible without departing from thespirit or scope thereof.

EXAMPLES

General Experimental Procedures

All solvents were distilled before use and were removed by rotaryevaporation at temperatures up to 35° C. Octadecyl functionalized silicagel (C18) was used for reversed-phase (RP) flash chromatography, andMerck silica gel 60, 200-400 mesh, 40-63 μm, was used for silica gelflash chromatography. Thin layer chromatography (TLC) was carried outusing Merck DC-plastikfolien Kieselgel 60 F₂₅₄, first visualized with aUV lamp, and then by dipping in a vanillin solution (1% vanillin, 1%H₂SO₄ in ethanol), and heating. Optical rotations were measured on aPerkin-Elmer 241 polarimeter. Mass spectra were recorded on a KratosMS-80 instrument. NMR spectra, at 25° C., were recorded at 500 or 300MHz for ¹H and 125 or 75 MHz for ¹³C on Varian INOVA-500 or VXR-300spectrometers. Chemical shifts are given in ppm on the delta scalereferenced to the solvent peaks CHCl₃ at 7.25 and CDCl at 77.0 ppm,(CH₃)₂CO at 2.15 and (CD₃)₂CO at 30.5 ppm, or CH₃OD at 3.30 and CD₃OD at39.0 ppm.

HPLC Conditions

The analytical HPLC equipment consisted of a Waters 717 autosampler, 600pump and controller, and a 2487 UV detector controlled by Omegasoftware. Samples were analyzed by using an RP-18 semi-preparativecolumn (Phenomenex Prodigy 5 mm C18 100A, 250×4.6 mm) with a guardcolumn (Phenomenex SecurityGuard cartridge containing a C18 ODS 4×3 mm,5 mm column) fitted at 30° C. Samples (5 ml) were analyzed using amobile phase flow rate of 5.0 ml/min, with UV detection at 280 nm.

Method 1

Time (minutes) CH₃CN H₂O containing 0.1% TFA 0 11 89 20 11 89 30 100 031 11 89 40 11 89Method 2

Time CH₃CN/H₂O (95:5) H₂O (minutes) containing 0.1% TFA containing 0.1%TFA 0 11 89 20 11 89 30 100 0 31 11 89 40 11 89

Example 1 3,4,3′,4′-Tetrahydroxybenzoin (Compound 1; DC-0001)Bis(3,4-methylenedioxy)benzoin (compound 1B; DC-0001B)

A solution of piperonal (5 g) in ethanol (6.5 ml) was treated with asolution of potassium cyanide (0.5 g) in water (5 ml), then refluxed for5 h. The resultant precipitate was filtered off, washed with water thencrystallized from ethanol to give DC-0001B (124 g, 45%) as an off whitecrystalline solid.

¹H-NMR(CDCl₃) 7.52 (1H, dd, J 2, 8 Hz), 7.39 (1H, d, J 2 Hz), 6.73-6.82(4H, m), 6.02 (2H, s), 5.91 (2H, m), 5.76 (1H, d, J 6 Hz) and 4.51 (1H,d, J 6 Hz).

M/z 287 ((M−CH)⁻, 100%).

Bis(3,4-methylenedioxy)benzil

A mixture of copper acetate (20 mg), ammonium nitrate (660 mg) andDC-0001B (2 g) in aq. acetic acid (80%, 10 ml) were refluxed togetherfor 90 minutes. The mixture was cooled then poured into water (100 ml)and the product extracted into ethyl acetate (2×100 ml), dried andevaporated in vacuo to give a yellow gum. Trituration from ethanol gavebis(3,4-methylenedioxy)benzil (1.35 g, 68%) as a pale yellow solid.

¹H-NMR 7.48 (2H, dd, J 2, 8 Hz) 7.47 (2H, d, J 2 Hz), 6.86 (2H, d, J 8Hz) and 6.08 (4H, s).

3,4,3′,4′-Tetrahydroxybenzil

To a stirred solution of bis(3,4-methylenedioxy)benzil (500 mg) in dryCH₂Cl₂ (50 ml) under nitrogen, was slowly added boron tribromide (1.6ml) then stirring continued for a further 3.5 hours. Methanol (100 ml)was added carefully, then the solvent evaporated in vacuo to a volume of1 ml, this addition and evaporation was repeated twice more. The productwas purified by column chromatography over silica gel when elution withdiethylether in dichloromethane gave 3,4,3′,4′-tetrahydroxybenzil (217mg, 47%) as a yellow powder.

¹H-NMR 9.35 (2H, bs), 8.80 (2H, bs), 7.48 (2H, d, J 2 Hz), 7.34 (2H, dd,J 2, 8 Hz) and 7.02 (2H, d, J 8 Hz).

M/z 273 ((M−H)⁺, 100%).

HPLC (method 2) 31.3 minutes.

3,4,3′,4″-Tetrahydroxybenzoin (Compound 1; DC-0001)

A solution of the tetrahydroxybenzil (200 mg) in methanol (20 ml) withpalladium hydroxide on carbon (20%, 10 mg) was stirred under anatmosphere of hydrogen for 5 minutes. The mixture was filtered throughCelite, and the solvents removed in vacua to give an orange gum.Separation by column chromatography over silica gel eluting with 20%ethyl acetate in dichloromethane gave DC-0001 as an off-white gum (55mg, 27%). Recrystallization from methanol/dichloromethane gave pureDC-0001 as an off-white powder (27 mg, 13%).

¹H-NMR ((CD₃)₂CO) 7.41 (1H, d, J 2 Hz), 7.35 (1H, dd, J 2, 8 Hz), 6.75(1H, d, J 8 Hz), 6.73 (1H, d, J 2 Hz), 6.69 (1H, d, J 8 Hz), 6.64 (1H,dd, J 2, 8 Hz), 5.69 (1H, bd) and 4.60 (1H, bd).

¹³C-NMR ((CD₃)₂CO) 198.22, 151.41, 145.77, 145.68, 145.43, 132.79,127.07, 123.92, 120.52, 116.69, 116.20, 115.59, 115.36 and 75.97.

M/Z 275 ((M−H)⁺, 100%).

HPLC (Method 1) 7.1 minutes.

Example 2 3,4,3′,4′-Tetrahydroxydiphenylmethane (compound 3; DC-0003)

Bis(3,4-methylenedioxyphenyl)methanol

To a solution of piperonal (0.75 g) in solution in dichloromethane (25ml) was added dropwise 3,4-(methylenedioxy)phenylmagnesium bromide (5ml, 1M solution in toluene/THF). The mixture was stirred at roomtemperature overnight, then poured onto water, extracted withdichloromethane, dried and evaporated in vacuo to give the crude alcoholas a brown gum. Purification by column chromatography over silica geleluting with ethyl acetate in CH₂Cl₂ (10 to 20%) gave the pure alcoholas a white gum (1.18 g, 87%).

¹H-NMR (CDCl₃) 6.7-6.8 (6H, m), 5.93 (4H, s), 5.66 (1H, bs) and 2.18(bs),

Bis(3,4-methylenedioxyphenyl)methane (compound 3B; DC-0003B)

A solution of the alcohol (2.61 g) in methanol (25 ml)/tetrahydrofuran(30 ml) was shaken with Pd(OH)₂/C(20%, 100 mg) under an atmosphere ofhydrogen for 12 hours. The mixture was filtered through Celite, then thesolvents removed in vacuo to give a brown gum (2.4 g). Crystallizationfrom acetone gave DC-0003B as white crystals (1.14 g, 44%).

¹H-NMR (CDCl₃) 6.6-6.8 (6H, m), 5.90 (4H, s) and 3.79(2H, s).

3,4,3′,4′-Tetrahydroxydiphenylmethane (compound 3; DC-0003)

To a stirred solution of DC-0003B (0.214 mg) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.4 ml) then stirring wascontinued for a further 3.5 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml; this was thenrepeated 2 more times. The product was purified by column chromatographyover silica gel when elution with ethyl acetate in dichloromethane gaveDC-0003 (48%) as an off-white solid.

¹H-NMR ((CD₃)₂CO) 7.73 (2H, s), 7.66 (2H, s), 6.74 (2H, d, J 8 Hz), 6.67(2H, d, J 2 Hz), 6.56 (2H, dd, J 2, 8 Hz) and 3.70 (2H, s).

¹³C-NMR ((CD₃)₂CO) 146.51, 144.80, 135.34, 121.59, 117.45, 116.64 and41.90.

M/z 232 (M ⁺, 100%).

HPLC (Method 1) 31.1 minutes.

Example 3 1,2-bis(3,4-dihydroxyphenyl)ethane (compound 4; DC-0004)

1,2-bis-(3,4-dihydroxyphenyl)ethane (compound 4; DC-0004)

A solution of the tetrahydroxybenzil (see Example 1) (70 mg) in methanol(10 ml) with palladium hydroxide on carbon (20%, 10 mg) was stirredunder an atmosphere of hydrogen for 2 hours, The mixture was filteredthrough Celite, and the solvents removed in vacuo to give an orange gum.Separation by column chromatography over silica gel eluting with 20%ethyl acetate in dichloromethane gave DC-0004 as an off white gum (43 g,68%).

¹H-NMR ((CD₃)₂CO) 7.73 (4H, bs), 6.80 (2H, d, J 8 Hz), 6,79 (2H, d, J 2Hz), 6.62 (2H, dd, J 2, 8 Hz) and 2.79 (4H, s).

M/z 245 (M−H)⁺, 100%).

HPLC (Method 2) 31.7 minutes.

Example 4 4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine(compound 8; DC-0008)

4,6-bis(3,4-methylenedioxyphenyl)-3-cyano-2-methylpyridine (compound 8B;DC-0008B)

To a solution of the chalcone (see below) (300 mg, 1.0 trunol) and3-aminocrotonitrile (82 mg, 1.2 mmol) in dry acetonitiile was addedpotassium tert-butoxide (560 mg) and the mixture stirred for 18 h. Themixture was then pouted into water, extracted with ethyl acetate, driedand evaporated in vacuo. Recrystallization from dichloromethane/ethergave DC-0008B (152 mg, 42%) as an off-white powder.

¹H-NMR(CDCl₃) 7.60 (2H, m), 7.52 (1H, s), 7.10 (2H, m), 6.93 (2H, m),6.07 (2H, s), 6.05 (2H, s) and 2.87 (3H, s).

M/z 359 ((M+1)⁺, 100%).

4,6-bis(3,4-dihydroxyphenyl)-3-cyano-2-methylpyridine (compound 8;DC-0008)

To a stirred solution of DC-0008B (0.10 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.2 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 this was thenrepeated 2 more times. The product was recrystallized frommethanol/acetone to give pure DC-0008 as small yellow crystals (64 mg,69%).

¹H-NMR ((CD₃)₂CO) 8.19 (1H, s), 7.86 (1H, d, J 2 Hz), 7.75 (1H, dd, J 2,8 Hz), 7.58 (1H, d, J 2 Hz), 7.45 (1H, dd, J 2, 8 Hz), 7.16 (1H, d, J 8Hz), 7.13 (1H, d, J 8 Hz), and 2.73 (3H, s).

M/z 335 ((M+1)⁺, 100%)

HPLC (method 2) 31.8 minutes.

Bis (3,4-methylenedioxy)chalcone (compound 6B; DC-0006B)

A mixture of piperonal (460 mg) and 3,4-methylenedioxyacetophenone (500mg) in ethanol (20 ml) was treated with 1M NaOH solution (4 ml), thenthe mixture was stirred overnight. The pale yellow crystalline solid wasfiltered off, washed with water then cold aqueous ethanol and dried togive pure bis(3,4-methylenedioxy)chalcone DC-0006B (476 mg, 53%).

¹H-NMR (CDCl₃) 7.72 (1H, d, J 16 Hz), 7.64 (1H, dd, J 2, 8 Hz), 752 (1H,d, J 2 Hz), 7.33 (1H, d, J 16 Hz), 7.16 (1H, d, J 2 Hz), 7.12 (1H, dd, J2, 8 Hz), 6.89 (1H d, J 8 Hz), 6.84 (1H, d, J 8 Hz), 6.06 (2H, s) and6.03 (2H, s).

M/z 297 ((M+1)⁺, 100%).

Example 5 1,4-bis(3,4-dihydroxybenzyl) piperazine (compound 9; DC-0009)

Method 1—Via Methylenedioxy-protected Compounds

1,4-bis-(3,4-methylenedioxybenzyl) piperazine (DC-0009B)

To a solution of piperazine (207 mg) in dry DMF (5 ml) under nitrogenwas added sodium hydride (80% w/w in oil, 250 mg), followed by3,4-methyleneciioxybenzylchloride (0.90 g) and the mixture stirred atroom temperature overnight, Aqueous NaOH (50 ml, 1M) was added slowly,then saturated NaCl solution (50 ml) and the product extracted withdichloromethane (2×100 ml). The organic layer was washed with water(2×100 ml), dried and evaporated in vacuo to give a white solid. Columnchromatography eluting with increasing proportions of ether indichloromethane gave pure DC-0009B (0.68 g, 80%) as a white powder.

¹H-NMR (CDCl₃) 6.85 (2H, s), 6.70 (4H, s), 5.94 (4H, s), 3.42 (4H, s)and 2.45 (8H, bs).

M/z 355 ((M+1)⁺, 100%).

1,4-bis-(3,4-dihydroxybenzyl) piperazine (DC-0009)

To a stirred solution of DC-0009B (200 mg) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.6 ml) then stirringcontinued for a further 30 minutes. Methanol (50 ml) was addedcarefully, then the solvent evaporated in vacua to a volume of 1 ml, andthis addition and evaporation was repeated twice more. Purification bycolumn chromatography over silica gel eluting with 20% methanol inchloroform gave a fraction containing crude product DC-0009 (51 mg, 27%)as a white powder.

¹H-NMR (CD₃)₂CO) 6.88 (2H, d, J 2 Hz), 6.78 (2H d, J 8 Hz), 6.67 (2H,dd, J 2, 8 Hz), 3.36 (4H, s) and 2.50 (8H, bs).

¹³C NMR (CD₃)₂CO) 146.50, 145.85, 131.17, 122.15, 117.78, 116.44, 63.72and 54.23.

M/z 331 ((M+H)⁺, 100%).

HPLC (Method 2) 3.79, 3.22 minutes for the mono and di protonated forms.

Method 2—Via Methoxy-protected Compounds

3,4-Dimethoxybenzyl chloride

3,4-dimethoxybenzyl alcohol (20 g, 119 mmol) was dissolved in toluene(60 ml) and cooled to 0° C. Thionyl chloride (7.48 g, 61.4 mmol) wasadded dropwise to the cooled solution of the alcohol over a period of 30minutes, and the reaction was maintained at 0° C. for an additional 30minutes. The reaction was quenched by pouring onto an ice/water mix (100ml), and the organic phase was separated. The aqueous phase was thenextracted into toluene (2×20 ml) and the combined toluene solution wasdried over anhydrous sodium sulfate. The toluene was removed at reducedpressure to afford an oil which solidified upon standing, with a yieldof 21 g. The material was characterized as a single spot by thin layerchromatography (TLC).

1,4-Bis(3,4-dimethoxybenzyl)piperazine

3,4-dimethoxybenzyl chloride (10 g, 53.6 mmol) was combined withpiperazine (2.3 g, 26.8 mmol) in anhydrous DMF (30 ml) and heated withstirring under nitrogen for 8 hours at 95-100° C. The cooled reactionmixture was diluted with water (100 ml) and acidified to pH 1 withconcentrated hydrochloric acid. The white precipitate was collected byfiltration and washed with water (50 ml). The solid was re-suspended inwater (50 ml) and the pH adjusted to >9 by the dropwise addition ofsodium hydroxide solution (50% NaOH in water). The resultant white solidwas collected by filtration and dried under vacuum at 50° C., yield 10g.

1,4-Bis(3,4-dihydroxybenzyl)piperazine (DC-0009)

1,4-Bis(3,4-dimethoxybenzyl)piperazine (5 g, 12.95 mmol) was combinedwith hydrobromic acid (50 ml of 48% w/w solution in water) and thesolution heated slowly over 1 hour to 145° C. Reaction was maintained at145° C. for 12 h at which time TLC revealed disappearance of startingmaterial. The cooled solution was diluted with water (200 ml), carefullyneutralized with saturated aqueous sodium hydrogen carbonate, and ethylacetate (100 ml) added. The crude aqueous solvent mixture was filteredthrough Celite and the ethyl acetate layer separated. The aqueous layerwas extracted with ethyl acetate (2×50 ml), and the combined extractswashed with water (50 ml), and dried (Na₂SO₄). The solvent was removedunder reduced pressure and the residue recrystallized from toluene andmethyl ethyl ketone to afford the product, DC-0009, 100 mg (98%, pure byHPLC analysis).

Example 6 N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane(compound 12; DC-0012)N,N′-bis(3,4-methylenedioxybenzyl)-trans-1,2-diaminocyclohexane(compound 12B; DC-0012B)

To a solution of piperonal (0.8 g, 5.3 mmol) and 1,2-diaminocyclohexane(0.296 g, 2.6 mmol) in dry methanol (25 ml) was added sodiumcyanoborohydride (0.38 g, 6 mmol) and the mixture stirred for 48 h. Themixture was filtered and the solvents removed in vacuo to give the crudeproduct. Crystallization from methanol gave DC-0012B as an off-whitecrystalline solid (0.298 g, 30%).

¹H-NMR(CDCl₃) 6.83 (2H, s), 6.75 (4H, s), 5.94 (4H, m), 3.80 (2H, d, j13 Hz), 3.56 (2H, d, J 13 Hz), 2.22 (2H, m), 2.18 (2H, m), 1.74 (4H, m),1.22 (2H, m) and 1.02 (2H, m).

N,N′-bis(3,4-dihydroxybenzyl)-trans-1,2-diaminocyclohexane (compound 12;DC-0012)

To a stirred solution of DC-0012B (0.25 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.31 ml), then stirring wascontinued for a further 4 hours. Methanol (100 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml; this additionand evaporation was then repeated twice more, and then water (2 ml) wasadded and the product lyophilized to give DC-0012 as a pale brown solid(150 mg, 64%).

¹H-NMR(D₂O) 6.88 (2H, br s), 6.84 (2H, d, J 8 Hz), 6.76 (2H, br d, J 8Hz), 4.20 (2H, d, J 13 Hz), 3.98 (2H, d, J 13 Hz), 3.41 (2H, br s), 2.24(2H, br s), 1.74 (2H, br s), 1.63 (2H, br and 1.40 (2H, br s).

M/z 359 ((M+1)⁺, 100%).

HPLC (Method 2) 8.2 minutes.

Example 7 2,4-bis(3,4-dihydroxybenzyl)-3-tropinone (compound 19;DC-0019)

A mixture of tropinone (418 mg, 3 mmol) and3,4-methylenedioxybenzaldehyde (900 mg, 6 mmol) in ethanol (20 m wastreated with 1M NaOH solution (4 ml), and then the mixture was stirredovernight. The yellow crystalline solid was filtered off, washed withwater, then cold aqueous ethanol, and dried to give pure DC-0019P (938mg, 77%).

¹H-NMR(CDCl₃) 7.73 (2H, s), 6.88 (6H, m), 6.02 (4H, s), 4.39 (2H, m),2.60 (2H, m), 2.31 (3H, s) and 1.98 (2H, q, J 8 Hz).

M/z 404 (M+1)⁺, 100%).

2,4-bis(3,4-methylenedioxybenzyl)-3-tropinone (compound 19B; DC-0019B)

A mixture of DC-0019P (500 mg, 1.24 mmol) and 10% Pd/C (100 mg) in ethylacetate (50 ml) was stirred overnight under an atmosphere of hydrogen.The mixture was filtered through Celite and evaporated in vacuo.Crystallization of the residue from dichloromethane/ether gave pureDC-0019B (366 mg, 72%) as a white crystalline solid.

¹H-NMR(CDCl₃) 6.69 (2H, d, J 8 Hz), 6.61 (2H, d, J 2 Hz), 6.58 (2H, dd,J 2, 8 Hz), 5.90 (4H, s), 3.17 (4H, m), 2.86 (2H, m), 2.36 (3H, s),2.24(2H, dd, J 8, 12 Hz), 1.83 (2H, m) and 1.60 (2H, q, J 8 Hz).

M/z 408 ((M+1)⁺, 100%).

2,4- bis(3,4-dihydroxybenzyl)-3-tropinone (compound 19; DC-0019)

To a stirred solution of DC-0019B (0.10 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.2 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml, this wasrepeated 2 more times. The product was crystallized from methanol togive pure DC-0019 (42 mg, 45%) as a white solid.

¹H-NMR (D₂O) 6.75 (2H, d, J 8 Hz), 6.68 (2H, d, J 2 Hz), 6.59 (2H, dd, J2, 8 Hz), 3.84 (2H, bs), 3.31 (4H, s), 3.07 (2H, dd, 6, 14 Hz), 2.82(311, s), 2.37 (dd, J 8, 14 Hz) and 2.05 (2H, d 8 Hz).

M/z 384 ((M+1)⁺, 100%).

HPLC (method 2) 30.9 minutes.

Example 8 α-(3,4-Dihydroxybenzamido)-3,4-dihydroxycinnamic acid3,4-dihydroxybenzylamide (compound 21; DC-0021)

2-(3,4-methylenedioxyphenyl)-4-(3,4-methylenedioxybenzylamino)methylene-4H-oxazol-5-one(DC-0021P)

DC-0021P is also referred to as DC-0022B, and is commercially available.It was prepared from (3,4-methylenedioxybenzoyl)aminoacetic acid[3,4-methylenedioxyhippuric acid] (prepared by the method of Acheson etal., J. Chem Soc. Abstracts, 1960:3457-3461, from3,4-methylenedioxybenzoic acid), by reaction with piperonaldehyde usingthe method described by Van der Eycken et al., Tet. Lett.,30(29):3873-3876, 1989.

¹H-NMR (CDCl₃) 8.09 (1H, d, J 2 Hz), 7.75 (1H, dd, J 2, 8 Hz), 7.62 (1H,d, J 2 Hz), 7.45 (1H, dd, J2, 8 Hz), 7.12 (1H, s), 6.94 (1H ,d, J 8 Hz),6.90 (1H, d, J 8 Hz), 6.11(2H, s) and 6.08 (2H, s).

m/z 338 (M+H)⁺.

α-(3,4-methylenedioxybenzamido)-3,4-methylenedioxycinnamic acid3,4-methylenedioxybenzyl-amide (compound 21B; DC-0021B)

A mixture of DC-0021P (250 mg, 0.74 mmol) and3,4-methylenedioxybenzylamine (0.112 g, 0.74 mmol) in acetic acid(glacial, 3 ml) were heated together under reflux for 30 minutes. Thereaction was quenched with ethyl acetate, washing with sodiumbicarbonate, dried and evaporated in vacua to give the crude product.Purification by column chromatography, eluting with hexane/ethyl acetate(50/50), followed by recrystallization from ethanol/water gave pureDC-0021B (218 mg, 60%).

¹H-NMR ((CD₃)₂CO) 9.09 (1H, bs), 8.06 (1H, bt, J 7 Hz), 7.70 (1H, dd, J2, 8 Hz), 7.56 (1H, d, J 2 Hz), 7.37 (1H, s), 7.16 (1H, d, J 2 Hz), 7.08(1H, dd, J 2, 8 Hz), 7.00 (1H, d, J 8 Hz), 6.94 (1H, d, J 2 Hz), 6.86(1H, d, J 8 Hz), 6.84 (1H, dd, J2, 8 Hz), 6.77 (1H, d, J 8 Hz), 6.14(2H, s), 6.02 (2H, s), 5.98 (2H, s) and 4.43 (2H, d, J 7 Hz).

M/z 489 ((M+1)⁺, 100%).

α-(3,4dihydroxybenzamido)-3,4-dihydroxycinnamic acid3,4-dihydroxybenzylamide (compound 21; DC-0021)

To a stirred solution of DC-0021B (85 mg) in dry CH₂Cl₂ (20 ml) undernitrogen, was slowly added boron tribromide (0.2 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml; this wasrepeated 2 more times. Purification by column chromatography over silicagel eluting with 20% methanol in chloroform gave pure DC-0021 as a paleyellow solid (42 mg, 53%).

¹H-NMR ((CD₃)₂CO) 7.75 (1H, d, J 2 Hz), 7.63 (1H, dd, J 2, 8 Hz), 7.50(1H, s), 7.34 (1H, d, J 2 Hz), 7.12 (1H, dd, J 2, 8 Hz), 7.00-7.04 (2H,m), 6.91 (1H, d, J 8 Hz), 6.80-6.85 (2H, m) and 4.68 (2H, s).

M/z 451 ((M −1)⁺, 100%).

HPLC (method 2) 27.1 minutes.

Example 9 1,4-bis(3,4-dihydroxybenzoyl)piperazine (compound 23; DC-0023)1,4-bis(3,4methylenedioxybenzoyl)piperazine (compound 23B; DC-0023B)

A suspension of piperonylic acid (0.5 g) in thionyl chloride (15 ml) wasrefluxed for 1 h under nitrogen, when a clear solution had been formed.The solvents were removed in vacuo to give the acid chloride as a whitesolid. The solid was dissolved in dry dichloromethane (7 ml) and addeddropwise to a stirred solution of piperazine (0.13 g) in drydichloromethane (20 ml) containing pyridine (0.5 ml). The mixture wasrefluxed for 30 minutes, diluted with more dichloromethane (50 ml), thenwashed with aqueous HCl (1M, 50 ml) followed by aqueous NaOH (1M, 50ml), dried and evaporated in vacuo to give the crude product.Crystallization from methanol/water gave DC-0023B as a white solid (532mg, 92%).

¹H-NMR (CDCl₃) 6.80-6.96 (6H, m), 6.00 (4H, s), and 3.62 (8H, bs).

1,4-bis(3,4-dihydroxybenzoyl)piperazine (compound 23; DC-0023)

To a stirred solution of DC-0023B (0.20 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.4 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml, this wasrepeated 2 more times. The product was crystallized frommethanol/dichloromethane to give pure DC-0023 (141 mg, 75%) as a whitesolid.

¹H-NMR (CD₃OD) 6.88 (2H, s), 6.81 (4H, s) and 3.66 (8H, s).

M/z 357 ((M−H)⁺, 100%).

Example 10 N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane(compound 26; DC-0026)N,N′-bis(3,4-methylenedioxybenzoyl)-trans-1,2-diaminocyclohexane(compound 26B; DC-0026B)

A suspension of piperonylic acid (0.5 g) in thionyl chloride (15 ml) wasrefluxed for 1 h under nitrogen, when a clear solution had been formed.The solvents were removed in vacuo to give the acid chloride as a whitesolid. The solid was dissolved in dry dichloromethane (7 ml) and addeddropwise to a stirred solution of trans-1,2-diaminocyclohexane (0.17 g)in dry dichloromethane (20 ml) containing pyridine (0.5 ml). The mixturewas refluxed for 30 minutes, diluted with more dichloromethane (50 ml),then washed with aqueous HCl (1M, 50 ml), followed by aqueous NaOH (1M,50 ml), dried and evaporated in vacuo to give the crude product.Crystallization from methanol/water gave DC-0026B as a white solid (544mg, 94%).

¹H-NMR (CDCl₃) 7.27 (2H, m), 6.77 (2H, d, J 8 Hz), 6.67 (2H, bs), 5.98(4H, s), 3.92 (2H, bs), 2.20 (2H, bd), 1.80 (2H, bs) and 1.38 (4H, bm).

N,N′-bis(3,4-dihydroxybenzoyl)-trans-1,2-diaminocyclohexane (compound26; DC-0026)

To a stirred solution of DC-0026B (0.20 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.4 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml, this additionand evaporation was repeated twice more. The product was crystallizedfrom methanol/dichloromethane to give pure DC-0026 (161 mg, 86%) as awhite solid.

¹H-NMR (CD₃OD) 7.18 (2H, s), 7.11 (2H, d, J 8 Hz), 6.73 (2H, d, J 8 Hz),3.89 (2H, m), 2.06 (2H, m), 1.83 (2H, m) and 1.44 (2H, m).

M/z 385 ((M−H)⁺, 100%).

HPLC (Method 1) 30.9 minutes.

Example 11 3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compound 51;DC-0051)

Method 1—Via Methylenedioxy-protected Compounds

3,4-methylenedioxybenzoic acid 3,4-methylenedioxyanilide (compound 51;DC-0051B)

To a solution of piperonylic acid (500 mg, 3 mmol) in dry CH₂Cl₂ (25 ml)under nitrogen, was added oxalyl chloride (573 mg, 4.5 mmol) with threedrops of dry DMF, and the mixture was stirred for 1 hour. Solvents wereremoved in vacuo giving the acid chloride as a white solid. To asolution of the acid chloride in dry CH₂Cl₂ (50 ml) under nitrogen,cooled to 0° C., was added dropwise, a solution made up of3,4-(methylenedioxy)aniline (498 mg, 30.1 mmol) and pyridine (0.5 ml) inCH₂Cl₂ (5 ml). The reaction mixture was stirred for 30 minutes at roomtemperature, then diluted by the addition of CH₂Cl₂ (100 ml), washedwith aqueous HCl (50 ml, 10%) and sodium bicarbonate solution (50 ml)then dried. Solvents were removed in vacuo to give the crude product asa brown crystalline material. Recrystallization from aqueous ethanolgave DC-0051B as small silvery crystals (0.516 g, 60%).

¹H-NMR(CDCl₃) 7.60 (1H, br s), 7.35 (3H, m), 6.88 (2H, m), 6.78 (1H, d,J 9 Hz), 6.06 (2H, s) and 5.98 (2H, s).

3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compound 51; DC-0051)

To a solution of DC-0051B (100 mg) in dry CH₂Cl₂ (25 ml) under nitrogenwas added BBr₃ (0.2 ml) and the mixture was stirred for 6 hours. Afterstirring, aqueous 3M HCl (25 ml) was carefully added to the reactionmixture. The product was extracted into EtOAc (200 ml), dried andevaporated in vacuo to give the crude product. Purification by columnchromatography (Silica: Hexane/EtOAc 30:70) gave DC-0051 as an off-whitesolid (71 mg, 77%).

¹H-NMR(CD₃OD) 7.60 (1H, br s), 7.38 (1H, d, J 2 Hz), 7.33 (1H, dd, J 2,8 Hz), 7.21 (1H, d, J 2 Hz), 6.89 (1H, dd, J 2, 8 Hz), 6.86 (1H, d, J 8Hz) and 6.76 (1H, d, J 8 Hz).

M/z 262 ((M+1)⁺, 100%)

HPLC (method 2) 15.1 minutes.

Method 2—Via Benzyloxy—and Methoxymethoxy-protected Compounds:

3,4-dibenzyloxybenzoyl chloride

3,4-dibenzyloxybenzoic acid (3.1 g. 9.3 mmol) was combined with pyridine(5 drops, catalytic) and thionyl chloride (15 ml, 205 mmol). Thesolution was heated at reflux for 4 h, cooled, and excess thionylchloride removed under reduced pressure. The crude product was dissolvedin benzene (50 ml), and stripped of solvent under vacuum. The benzoylchloride (theoretical yield 3.4 g) was then dissolved in dichloromethaneand used directly in the next step.

3,4- dibenzyloxybenzoic acid 3,4-di(methoxymethoxy)anilide

3,4-di(methoxymethoxy)aniline (0.484 g, 22 mmol) was dissolved indichloromethane (5 ml) and pyridine (3 ml) and cooled to −5° C., whilestirring under nitrogen. A solution of 3,4-dibenzyloxybenzoyl chloridein dichloromethane (0.8 g, 2.2 mmol of acid chloride) was added dropwiseover 30 minutes. The reaction was allowed to stir at 0° C. for 30minutes then warmed to room temperature over 30 minutes. The reactionwas diluted with dichloromethane (100 ml), washed with aqueous citricacid (3×300 ml of a 2% w/v solution), aqueous sodium hydroxide (2×35 mlof a 2% w/v solution) and dried(Na₂SO₄). Removal of the solvent underreduced pressure afforded a solid, 0.97 g. The crude product wastriturated with warm methanol (10 ml) and filtered to afford the desiredproduct, 0.5 g.

3,4-dihydroxybenzoic acid 3,4-di(methoxymethoxy)anilide

3,4-dibenzyloxybenzoic acid 3,4-di(methoxymethoxy)benzanilide (0.2 g,0.4 mmol) was combined with ethanol (10 ml), and palladium on charcoal(40 mg of 10% Pd/C). The reaction was heated to reflux with stirringunder nitrogen, and ammonium formate (0.8 g, 12.7 mmol) was addedportion wise over 15 min and then held at reflux for two hours. Thecooled reaction solution was filtered to remove the catalyst andconcentrated under reduced pressure to afford the crude product, 0.13 g.

3,4-dihydroxybenzoic acid 3,4-dihydroxyanilide (compound 51; DC-0051)

3,4-dihydroxybenzoic acid 3,4-di(methoxymethoxy)benzanilide (0.17 g,0.49 mmol) was combined with a 25% solution of hydrogen chloride inisopropyl alcohol (15 ml) and water (1 ml). The reaction was stirred atroom temperature for 1 h and the solvent removed under reduced pressure.Trituration with diethyl ether (5 ml) afforded DC-0051 as a solid whichwas dried under vacuum at 30° C., yield 60 mg.

Example 12 3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide (compound52; DC-0052)

3,4-methylenedioxybenzoic acid 3,4-methylenedioxybenzylamide (compound52B; DC-0052B)

A suspension of piperonylic acid (0.5 g) in thionyl chloride (15 ml) wasrefluxed for 1 h under nitrogen, when a clear solution had been formed.The solvents were removed in vacuo to give the acid chloride as a whitesolid. The solid was dissolved in dry dichloromethane (7 ml) and addeddropwise to a stirred solution of piperonylamine (0.45 g) in drydichloromethane (20 ml) containing pyridine (0.5 ml). The mixture wasrefluxed for 30 minutes, diluted with more dichloromethane (50 ml), thenwashed with aqueous HCl (1M, 50 ml) followed by aqueous NaOH (1M, 50ml), dried and evaporated in vacuo to give the crude product.Crystallization from methanol/water gave DC-0052B as a white solid (733mg, 79%).

¹H-NMR (CDCl₃) 7.27 (2H, m), 6.79 (4H, m), 6.01 (2H, s), 5.94 (2H, s)and 4.51 (2H, d, J 5 Hz).

3,4-dihydroxybenzoic acid 3,4-dihydroxybenzylamide (compound 52;DC-0052)

To a stirred solution of DC-0052B (0.20 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.4 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml; this was thenrepeated 2 more times. The product was crystallized frommethanol/dichloromethane to give pure DC-0052 (65 mg, 35%) as a whitesolid.

¹H-NMR (CD₃OD) 7.29 (2H, s), 7.22 (2H, d, J 8 Hz), 6.78 (4H, m), 6.67(4H, m) and 4.38 (4H, d, J 5 Hz).

M/z 274 ((M−H)⁺, 100%)

HPLC (Method 1) 10.4 minutes.

Example 13 3-(3,4-dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide(compound 57; DC-0057)

3-(3,4-methylenedioxyphenyl) propionic acid 3,4-methylenedioxyanilide(compound 57B; DC-0057B)

To a solution of 3,4-(methylenedioxy)dihydrocinnamic acid (0.4 g) in dryCH₂Cl₂ (25 ml) under nitrogen, was added oxalyl chloride (0.5 ml) withthree drops of dry DMF and the mixture stirred for 1 hour. Solvents wereremoved in vacuo giving the acid chloride as a yellow solid. To asolution of the acid chloride in dry CH₂Cl₂ (50 ml) under nitrogen,cooled to 0° C., was added dropwise, a solution of3,4-(methylenedioxy)aniline (0.35 g) and pyridine (0.2 ml) in CH₂Cl₂ (5ml). The reaction mixture was stirred for 30 minutes at roomtemperature, diluted with CH₂Cl₂ (100 ml), washed with aqueous HCl (100ml, 10%) and sodium bicarbonate solution (100 ml) then dried andevaporated in vacuo to give DC-0057B as a dark brown powder (0349 g,85%).

¹H-NMR (CDCl₃) 7.15 (1H, d, J 2 Hz), 6.86 (1H, bs), 6.60-6.75 (5H, m),5.93 (2H, s), 5.92 (2H, s), 2.95 (2H, t, J 4 Hz) and 2.57 (2H, t, J 4Hz).

3-(3,4- dihydroxyphenyl)propionic acid 3,4-dihydroxyanilide (compound57; DC-0057)

To a stirred solution of DC-0057B (0.20 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.4 ml), then stirring wascontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacua to a volume of 1 ml, this wasrepeated 2 more times to give pure DC-0057 as a brown solid (143 mg,77%).

¹H-NMR ((CD₃)₂CO) 7.31 (1H, s), 6.98 (3H, m), 6.84 (1H, d, J 8 Hz), 6.78(1H, dd, J 2, 8 Hz), 3.24 (2H, m) and 3.16 (2H, m).

M/z 370, 368 (M+HBr)⁺, 288 ((M−H)⁺, 100%)

HPLC (Method 2) 20.6 minutes.

Example 14 3-(3,4-dihydroxyphenyl)propionic acid3,4-dihydroxybenzylamide (compound 58; DC-0058)

3-(3,4-methylenedioxyphenyl)propionic acid 3,4-methylenedioxybenzylamide(compound 58B; DC-0058B)

To a solution of 3,4-methylenedioxydihydrocinnamic acid (0.4 g) in dryCH₂Cl₂ (25 ml) under nitrogen, was added oxalyl chloride (0.5 ml) withthree drops of dry DMF and the mixture was stirred for 1 hour. Solventswere removed in vacuo giving the acid chloride as a yellow solid. To asolution of the acid chloride in dry CH₂Cl₂ (50 ml) under nitrogen,cooled to 0° C., was added dropwise, a solution of3,4-(methylenedioxy)benzylamine (0.35 g) and pyridine (0.2 ml) in CH₂Cl₂(5 ml). The reaction mixture was stirred for 30 minutes at momtemperature, diluted with CH₂Cl₂ (100 ml), washed with aqueous HCl (100ml; 10%) and sodium bicarbonate solution (100 ml) then dried andevaporated in vacua to give DC-0058B as an off white powder (0.536 g,80%).

3-(3,4-dihydroxyphenyl)propionic acid 3,4dihydroxybenzylamide (compound58; DC-0058)

To a stirred solution of DC-0058B (0.20 g) in dry CH₂Cl₂ (25 ml) undernitrogen, was slowly added boron tribromide (0.4 ml), then stirring wascontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml; this wasrepeated 2 more times to give pure DC-0058 as a brown solid (143 mg,77%).

¹H-NMR ((CD₃)₂CO) 9.62 (1H, bs), 6.95 (1H, d, J 2 Hz), 6.91 (1H, d, J 2Hz), 6.88 (1H, d, J 8 Hz), 6.83 (1H, d, J 8 Hz), 6.67 (2H, m), 6.35 (4H,bs) 4.47 (2H, s) and 3.00 (4H, m).

M/z 302 ((M−H)⁺, 100%)

HPLC (Method 2) 19.4 minutes.

Example 15 3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide (compound61; DC-0061)

3,4-methylenedioxycinnamic acid 3,4-methylenedioxybenzylamide (compound61B; DC-0061B)

To a solution of 3,4-methylenedioxycinnamic acid (0.5 g, 2.6 mmol) indry CH₂Cl₂ (25 ml) under nitrogen, was added oxalyl chloride (0.33 g,2.6 mmol) with three drops of dry DMF and the mixture was stirred for 1hour. Solvents were removed in vacuo giving the acid chloride as ayellow solid. To a solution of the acid chloride in dry CH₂Cl₂ (50 ml)under nitrogen, cooled to 0° C., was added dropwise, a solution of3,4-(methylenedioxy)benzylamine (0.393 g, 2.6 mmol) and pyridine (0.205g, 2.6 mmol in CH₂Cl₂ (5 ml). The reaction mixture was stirred for 30minutes at room temperature, diluted with CH₂Cl₂ (100 ml), washed withaqueous HCl (100 ml, 10%) and sodium bicarbonate solution (100 ml) thendried and evaporated in vacuo to give DC-0061B as a dull yellow powder(0.523 g, 62%).

¹H-NMR(CDCl₃) 7.58 (1H, d, J 16 Hz), 6.98 (2H, m), 6.70-6.84 (4H, m),6.22 (1H, d, J 16 Hz), 6.00 (2H, s), 5.96 (2H, s) and 4.47 (2H, d, J 6Hz).

M/z 326 ((M+1)⁺, 100%)

3,4-dihydroxycinnamic acid 3,4-dihydroxybenzylamide (compound 61;DC-0061)

To a stirred solution of DC-0061B (0.3 g, 0.94 mmol) dissolved in dryCH₂Cl₂ (25 ml) was slowly added boron tribromide (1.16 g, 4.6 mmol),then stirring continued for a further 12 hours. Dilute HCl (25 ml) wascarefully added, then 200 ml of water, and the product was extractedinto ethyl acetate (2×100 ml), dried and evaporated in vacuo to give thecrude product. Purification by column chromatography eluting withhexane/ethyl acetate (1:4) gave DC-0061 as an off-white solid (36 mg,13%).

¹H-NMR ((CD₃)₂CO) 7.54 (1H, d, J 16 Hz), 7.12 (1H, d, J 2 Hz), 6.96 (1H,dd, J 2, 8 Hz), 6.85-6.94 (2H, m), 6.80 (1H, d, J 8 Hz), 6.70 (1H, dd, J2,8 Hz), 6.58 (1H, d, J 16 Hz) and 4.41 (2H, s).

M/z 300 ((M−1)⁺, 100%)

HPLC (method 2) 30.0 minutes.

Example 16 Oxalic acid bis(3,4-dihydroxyanilide) (compound 63; DC-0063)

Method 1—Via Methylenedioxy-protected Compounds

Oxalic acid bis(3,4-methylenedioxyanilide) (compound 63B; DC-0063B)

To a solution of oxalyl chloride (165 mg, 1.3 mmol) in dry CH₂Cl₂ (50ml) under nitrogen, cooled to 0° C., was added dropwise, a solution of3,4-(methylenedioxy)aniline (400 mg, 2.92 mmol) and pyridine (230 mg,2.92 mmol) dissolved in dry CH₂Cl₂ (50 ml). The reaction mixture wasstirred for further 30 min at room temperature, then washed with diluteaqueous HCl (50 ml). The organic layer was separated, dried andevaporated in vacuo to give DC-0063B as a gray powder (0.351 g, 82%).

¹H-NMR (CDCl₃) 10.78 (2H, s), 7.53 (2H, d, J 2 Hz), 739 (2H, dd, J 2, 8Hz), 6.96 (2H, d, J 8 Hz) and 6.06 (4H, s).

Oxalic acid bis(3,4-dihydroxyanilide) (compound 63: DC-0063)

To a stirred solution of DC-0063B (0.3 g, 0.91 mmol) dissolved in dryCH₂Cl₂ (25 ml) was slowly added boron tribromide (1.14 g, 4.7 mmol) thenstirring continued for a further 4 hours. Dilute HCl (25 ml) wascarefully added, then water (200 ml) and the product extracted intoethyl acetate (2×200 ml), dried and evaporated in vacuo to give thecrude product. The crude product was dissolved in acetone (25 ml) andfiltered. The acetone was evaporated in vacuo to give DC-0063 as anoff-white solid (171 mg, 62%).

¹H-NMR((CD₃)₂CO) 9.72 (2H, br s), 8.05 (2H, br s), 7.89 (2H, br s), 7.52(2H, d, J 2 Hz), 7.20 (2H, dd, J 2, 8 Hz) and 6.83 (2H, d, J 8 Hz).

M/z 303 ((M−1)⁺, 100%)

HPLC (method 2) 29.1 minutes.

Method 2—Via Methoxymethoxy-protected Compounds:

Oxalic acid bis(3,4-di(methoxymethoxy)anilide)

3,4-di(methoxymethoxy)aniline (1.5 g, 7 mmol) was dissolved indichloromethane (50 ml) and cooled to 0° C., while stirring undernitrogen. Pyridine (3.75 ml, 46 mmol) was added followed by dropwiseaddition of oxalyl chloride (0.4 g, 3.5 mmol) in dichloromethane (5 ml)over 20 minutes. The reaction was stirred for a further 10 min andallowed to warm to room temperature. The suspension was filtered. Theresidue was washed with hexane (5 ml) to remove excess pyridine. Thecrude product was triturated with methanol (5 ml) and filtered to affordthe pure protected anilide, 420 mg.

Oxalic acid bis(3,4dihydroxyanilide)

Oxalic acid bis(3,4-di(methoxymethoxy)anilide) (0.17 g, 0.36 mmol) wascombined with a 25% solution of hydrogen chloride in isopropyl alcohol(1.7 4. The reaction was stirred at room temperature overnight, and thesolvent was removed under reduced pressure. Trituration with diethylether (5 ml) afforded DC-0063, 60 mgs.

Example 17 Succinic acid bis(3,4-dihydroxyanilide) (compound 66;DC-0066)

Method 1—Via Methylenedioxy-protected Compounds

Succinic acid bis(3,4-methylenedioxyanilide) (compound 66B; DC-0066B)

To a suspension of succinic acid (200 mg, 1.7 mmol) in dry CH₂Cl₂ (25ml) under nitrogen was added oxalyl chloride (645 mg, 5.08 mmol) withthree drops of dry DMF, and the mixture was stirred for 1 hour. Solventswere removed in vacuo giving the acid chloride as a yellowish solid. Toa stirred solution of 3,4-(methylenedioxy)aniline (582 mg, 4.25 mmol)and pyridine (400 mg, 5.08 mmol) in dry CH2Cl₂ (50 ml) under nitrogen at0° C. was added drop-wise a solution of the acid chloride in dry CH₂Cl₂(25 ml) and stirred for a further 2 hours. The solvents were removed invacuo to give the crude product. The crude material was resuspended inEtOAc (250 ml) then washed with dilute aqueous HCl (2×150 ml), saturatedsodium bicarbonate (2×150 ml) and water (1×150 ml). The EtOAc was thenremoved by rotary evaporation. The product was scooped out onto filterpaper and washed with water and allowed to dry to give DC-0066B as awhite solid (514 mg, 78%).

¹H-NMR(CDCl₃) 9.97 (2H, s), 7.34 (2H, d, J 2 Hz), 6.99 (2H, dd, J 2, 8Hz), 6.86 (2H, d, J 8 Hz), 6.00 (4H, s) and 2.63 (4H, s).

Succinic acid bis(3,4-dihydroxyanilide) (compound 66; DC-0066)

To a stirred solution of DC-0066B (0.3 g, 0.78 mmol) in dry CH₂Cl₂ (25ml) was slowly added BBr₃ (0.978 g, 3.9 mmol) then stirring continuedfor a further 4 hours. Dilute HCl (25 ml) was carefully added, then 200ml of water and the product extracted into ethyl acetate (2×100 ml),dried and evaporated in vacuo to give DC-0066 as an off white solid (97mg, 35%).

¹H-NMR ((CD₃)2CO) 8.88 (2H, br s), 7.83 (2H, br s), 737 (2H, br s), 7.34(2H, d, J 2 Hz), 6.90 (2H, dd, J 2, 8 Hz), 6.71 (2H, d, J 8 Hz) and 2.65(4H, s).

M/z 331 ((M−1)⁺, 100%)

HPLC (method 2) 10.6 minutes.

Method 2—Via Methoxymethoxy-protected Compounds:

Succinic acid bis(3,4-di(methoxymethoxy)anilide)

3,4-di(methoxymethoxy)aniline (1 g, 4.7 mmol) was dissolved indichloromethane (25 ml) and cooled to 0° C., while stirring undernitrogen. Pyridine (1 ml, 12 mmol) was added followed by dropwiseaddition of succinyl chloride (0.35 g, 2.3 mmol) in dichloromethane (10ml) over 20 minutes. The reaction was stirred for a further 2 hours andallowed to warm to room temperature. The suspension was filtered, andthe white solid collected washed with hexane (10 ml) and then methanol(4 ml) to afford the anilide, 350 mg.

Succinic acid bis(3,4-dihydroxyanilide) (compound 66; DC-0066)

Succinic acid bis(3,4-di(methoxymethoxy)anilide) (0.15 g, 0.3 mmol) wascombined with a 25% solution of hydrogen chloride in isopropyl alcohol(1.5 ml) and water (1.5 ml). The reaction was stirred at roomtemperature for 3 h and the solvent was removed under reduced pressure.Trituration with diethyl ether afforded DC-0066 as a solid which wasdried under vacuum at 30° C., yield 60 mg.

Example 18 Succinic acid bis(3,4-dihydroxybenzylamide) (compound 67;DC-0067)

Method 1—Via Methylenedioxy-protected Compounds

Succinic acid bis(3,4-methylenedioxybenzylamide) (compound 67B;DC-0067B)

To a solution of succinic acid (200 mg, 1.7 mmol) in dry CH₂Cl₂ (25 ml)under nitrogen, was added oxalyl chloride (645 mg, 5.1 mmol) with threedrops of dry DMF and the mixture was stirred for 1 hour. Solvents wereremoved in vacuo giving the acid chloride as a yellow solid. To asolution of the acid chloride in dry CH₂Cl₂ (50 ml) under nitrogen,cooled to 0° C., was added drop-wise, a solution of3,4-methylenedioxybenzylamine (634 mg, 4.2 mmol) and pyridine (0.33 ml)in CH₂CL₂ (50 ml). The reaction mixture was stirred for a further 2hours at room temperature, then the solvents removed in vacuo to givethe crude product. The crude material was resuspended in EtOAc (250 ml)then washed with dilute aqueous HCl (2×150 ml), saturated sodiumbicarbonate (2×150 ml) and water (1×150 ml). The EtOAc was evaporated invacuo. Recrystallization from ethanol and water gave DC-0067B as whiteflaky crystals (275 mg, 42%).

¹H-NMR (DMSO-d₆) 8.31 (2H, t, J 6 Hz), 6.85 (4H, m), 6.74 (2H, dd, J 2,8 Hz), 6.01 (4H, s), 4.19 (4H, d, J 6 Hz) and 2.42 (4H, s).

Succinic acid bis(3,4-dihydroxybenzylamide) (compound 67; DC-0067)

To a stirred solution of DC-0067B (0.25 g, 0.65 mmol) dissolved in dryCH₂Cl₂ (25 ml) was slowly added boron tribromide (0.81 g, 0.31 ml), thenstirring continued for a further 4 hours. Dilute HCl (25 ml) wascarefully added, then brine (125 ml) and the product extracted intoethyl acetate (2×100 ml), dried and evaporated in vacuo to give DC-0067as an off-white solid (180 mg, 77%).

¹H-NMR((CD₃)₂CO) 7.90 (2H, br s), 7.74 (2H, br s), 7.42 (2H, br s), 6.79(2H, d, J 2 Hz), 6.77 (2H, d, J 8 Hz), 6.62 (2H, dd, J 2, 8 Hz), 4.22(4H, d, J 7 Hz) and 2.53 (4H, s).

M/z 359 ((M−1), 100%).

HPLC (method 2) 12.3 minutes.

Method 2—Via Benzyloxy-protected Compounds:

Succinic acid bis(3,4-dibenzyloxybenzylamide)

3,4-dibenzyloxybenzylamine (1.1 g, 3.45 mmol) was dissolved in anhydrouspyridine (8 ml) and cooled to 0° C. with stirring under nitrogen. Tothis solution, succinyl chloride (0.23 g, 1.42 mmol) was added dropwiseover 30 minutes as a solution in dichloromethane (50 ml), whilemaintaining the reaction mixture at 0° C. The reaction was allowed towarm to room temperature and stirred for an additional 45 minutes. Thereaction was poured onto crushed ice (70 g) and the dichloromethanelayer was separated. The organic extract was washed with dilute aqueoushydrochloric acid (2×20 ml of 0.1M solution), water (20 ml), and dried(Na₂SO₄). Removal of the solvent at reduced pressure afforded a crudesolid, which was triturated with methanol (5 ml) to afford afterfiltration the protected diamide, yield 300 mg.

Succinic acid bis(3,4-dihydroxybenzylamide) (compound 67; DC-0067)

Succinic acid bis(3,4-dibenzyloxybenzylamide) (300 mg, 0.42 mmol) wasdissolved in THF (50 ml) in a pressure bottle and warmed to 35 C toensure dissolution of the solid. Palladium on carbon (50 mg 10% Pd/C)was added, and the vessel was pressurized with hydrogen (to 3 atm). Thereaction was agitated for 1 hour at mom temperature, whereupon TLCrevealed reaction had gone to completion. The catalyst was removed byfiltration, and the solvent removed under reduced pressure to affordDC-0067 as a crude solid (20 mg). This material was recrystallized fromtoluene and methanol to afford DC-0067.

Example 19 Bis(3,4-dihydroxybenzyl)amine (compound 73; DC-0073)Bis(3,4-dimethoxybenzyl)amine

To a solution of 3,4-dimethoxybenzaldelyde (1 g, 6 mmol) in anhydrousmethanol (10 ml) was added 3,4-dimethoxybenzylamine (1 g, 5.9 mmol) andthe solution stirred under nitrogen at mom temperature for 3 hours. Themethanol was removed under reduced pressure to afford the crude imine,1.9 g. The imine was dissolved in THF (10 ml) and acetic acid (4 ml),and sodium cyanoborohydride (0.38 g, 6 mmol) was added portionwise over30 minutes. The solution was stirred for an additional 30 minutes atroom temperature, and the solvents were removed under reduced pressure.The residue was neutralized with saturated aqueous sodium hydrogencarbonate, and the solid crude product was isolated by filtration, anddried under vacuum at 50° C. overnight, yield 0.6 g.

Bis(3,4-dihydroxybenzyl)amine (compound 73; DC-0073)

The crude bis(3,4-dimethoxybenzyl)amine (0.6 g) was combined withhydrobromic acid (6 ml of 48% w/w solution in water) and slowly heatedwith stirring, to 145° C. over 1 h. The reaction was maintained at 145°C. for 12 h, allowed to cool to room temperature, and poured into water(25 ml). The reaction mixture was neutralized with saturated aqueoussodium hydrogen carbonate, and extracted with ethyl acetate (25 ml). Theorganic layer was washed into water (2×25 ml), dried (Na₂SO₄) and thesolvent removed under reduced pressure to afford DC-0073 as a solid, 160mg.

Example 20 Tris(3,4-dihydroxybenzyl)amine (compound 75; DC-0075)

Tris(3,4-methylenedioxybenzyl)amine (compound 75B; DC-0075B)

To a stirred solution of piperonal (0.9 g, 6 mmol) and ammonium acetate(0.15 g, 2 mmol) in acetonitrile (25 ml) was added sodiumcyanoborohydride (0.44 g, 7 mmol) and the mixture was stirred for 4days. The solvent was removed in vacuo, then the residue dissolved indichloromethane (100 ml) and washed with sat. sodium bicarbonate, driedand the solvent removed in vacuo to give a brown gum. Purification bycolumn chromatography over silica gel eluting with 50% dichloromethanein hexane gave the pure DC-0075B as a pale brown gum (135 mg, 5%).

¹H-NMR (CDCl₃) 6.91 (3H, m), 6.73-6.80 (6H, m), 5.94 s) and 3.42 (2H, m)

M/z 420 ((M+1)+, 100%).

Tris(3,4-dihydroxybenzyl)amine (compound 75; DC-0075)

To a stirred solution of DC-0075B (135 mg) in dry CH₂Cl₂ (20 ml) undernitrogen, was slowly added boron tribromide (0.2 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml, this additionand evaporation was repeated twice more. Purification by columnchromatography over silica gel eluting with 20% methanol in chloroformgave mostly pure DC-0075 (72 mg, 58%) as a pale brown gum. PreparativeHPLC then gave the pure DC-0075 as a white gum (26 mg, 21%).

¹H-NMR (CD₃OD) 6.82-6.86 (2H, m), 6.74 (1H, dd, J 2, 8 Hz) and 4.07 (2H,s).

M/z 384 ((M+1)+, 100%).

HPLC (method 2) 12.3 minutes.

Example 21 1,3-Bis(3,4-dihydroxyphenyl)urea (compound 76; DC-0076)

1,3-Bis(3,4-methylenedioxyphenyl)urea (compound 67B; DC-0076B)

A solution of 3,4-methylenedioxyaniline (0.35 g) and3,4-methylenedioxyphenyl isocyanate (0.4 g) in benzene (25 ml) wasrefluxed for 1 hour. The precipitate formed was filtered, washed withbenzene then dried to give pure DC-0076B (0.697 g, 95%) as a pale brownsolid.

¹H-NMR (CDCl₃/(CD₃)₂CO) 7.35 (2H, bs), 6.93 (2H, s), 6.45 (4H, s) and5.67 (4H, s).

1,3-Bis(3,4-dihydroxyphenyl)urea (compound 76; DC-0076)

To a stirred solution of DC-0076B (150 mg) in dry CH₂Cl₂ (20 ml) undernitrogen, was slowly added boron tribromide (0.2 ml) then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml, and thisaddition and evaporation was repeated twice more. Purification by columnchromatography over silica gel eluting with 20% methanol in chloroformgave pure DC-0076 (113 mg, 82%) as a pale brown solid.

¹H-NMR (D₂O/(CD₃)CO) 7.09 (2H, d, J 2Hz), 6.76 (2H, d, J 8 Hz) and 6.70(2H, dd, J 2, 8 Hz),

M/z 551 ((2M-H)+, 100%), 275 ((M-H)+, 85%).

HPLC (Method 2) 5.8 min.

Example 22 1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxybenzyl)urea (DC-4077)

1-(3,4-methylenedioxyphenyl)-3-(3,4-methylenedioxybenzyl)urea (DC-0077B)

A solution of 3,4-methylenedioxybenzylamine (0.37 g) and3,4-methylenedioxyphenyl isocyanate (0.4 g) in benzene (25 ml) wasrefluxed for 1 hour. The precipitate formed was filtered, washed withbenzene then dried to give pure DC-0077B (0.78 g, 98%) as a pale brownsolid.

¹H NMR (CDCl) 8.42 (1H, s, NH), 7.21 (1H, d, J 2 Hz), 6.88 (2H, m), 6.79(2H, m), 6.71 (1H, dd, J 2, 8 Hz), 6.49 (1H, t, J 6 Hz, NH), 6.01 (2H,s), 5.97 (2H, s) and 4.21 (2H, d, J 6 Hz).

M/z 315 ((M+1)+, 100%).

1-(3,4-dihydroxyphenyl)-3-(3,4-dibydroxybenzyl)urea (DC-0077)

To a stirred solution of DC-0077B (200 mg) in dry CH₂Cl₂ (50 ml) undernitrogen, was slowly added boron tribromide (0.4 ml) then stirringcontinued for a further 3 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml, this wasrepeated 2 more times. Purification by column chromatography over silicagel eluting with 20% methanol in chloroform gave a fraction containingcrude product. Preparative HPLC gave pure DC-0077 (19 mg, 11%) as a palebrown solid.

¹H NMR (D₂O) 6.55-6.80 (6H, m) and 4.12 (2H, s).

M/z 290 ((M)+, 100%).

HPLC (method 2) 12.7 min.

Example 23 1-(3,4-Dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea(compound 78; DC-0078)

1-(3,4-methylenedioxyphenyl)-3-(3,4-methylenedioxyphenethyl)urea(compound 78B; DC-0078B)

A solution of 3,4-methylenedioxyphenylethylamine (0.25 g, 1.5 mmol) and3,4-methylenedioxyphenyl isocyanate (0.25 g, 1.5 mmol) in benzene (25ml) was refluxed for 1 hour. The precipitate formed was filtered, washedwith benzene then dried to give pure DC-0078B (0.43 g, 85%) as a palebrown solid.

¹H-NMR((CD₃)₂CO) 7.83 (1H, bs), 7.31 (1H, d, J 2 Hz), 6.72-6.82 (5H, m),5.99 (2H, s), 5.95 (2H, s), 5.68 (1H, bt, J 7 Hz), 3.44 (2H, q, J 7 Hz),and 2.74 (2H, t, J 7 Hz).

M/z 327 ((M-1)+, 100%),

1-(3,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenethyl)urea (compound 78:DC-0078)

To a stirred solution of DC-0078B (105 mg) in city CH₂Cl₂ (20 ml) undernitrogen, was slowly added boron tribromide (0.2 ml), then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml; this additionand evaporation was repeated twice more. Purification by columnchromatography over silica gel eluting with 20% methanol in chloroformgave pure DC-0078 (78 mg, 80%) as a pale brown solid.

¹H-NMR ((CD₃)₂CO) 6.97 (2H, m), 6.86-6.89 (3H, m), 6.68 (1H, dd, J 2, 8Hz), 3.66 (2H, t, J 7 Hz), and 2,87 (2H, t, J 7 Hz).

M/z 303 ((M-1)+, 100%).

HPLC (method 2) 33.7 min.

Example 24 Dibenzo[c,f][2,7]naphthyridine-2,3,10,11-tetraol (compound85; DC-0085)

2,3,10,11-Tetramethoxydibenzo[c,f][2,7]naphthyridine (DC-0085P)

DC-0085P was prepared as described by Upton et al. J. Pharm Pharmacol,50(5):475-482, 1998. Veratrole was reacted with veratric acid to givethe protected benzophenone, which was nitrated to give the dinitrocompound, and this was reduced to the diamine by treatment with tin inhydrochloric acid and acetic acid. The diamine was isolated, and thencondensed with malonaldehyde bis(dimethyl acetal) to give DC-0085P.

Dibenzo[cf][2,7]naphthyridine-2,3,10,11-tetraol (DC-0085)

To a stirred solution of DC-0085P (100 mg) in dry CH₂Cl₂ (20 ml) undernitrogen, was slowly added boron tribromide (0.2 ml), then stirringcontinued for a further 2 hours. Methanol (50 ml) was added carefully,then the solvent evaporated in vacuo to a volume of 1 ml, and thisaddition and evaporation was repeated twice more. Purification bycrystallization from methanol/chloroform gave DC-0085 (36 mg, 38%) as anorange crystalline solid.

¹H-NMR (CD₃OD) 9.63 (2H, s), 8.63 (2H, s) and 7.64 (2H, s).

M/z 296 (M+2)+, 100%)

HPLC (method 1) 24.3 min.

Example 25 Compounds of this Invention are Potent Disrupters ofAlzheimer's Aβ 1-42 Fibrils

The compounds prepared in the preceding Examples were found mostly to bepotent disruptors/inhibitors of Alzheimer's disease β-amyloid protein(Aβ) fibrils. In a set of studies, the efficacy of the compounds tocause a disassembly/disruption of pre-formed amyloid fibrils ofAlzheimer's disease (i.e. consisting of Aβ 1-42 fibrils) was analyzed.

Part A—Thioflavin T Fluorometry Data

In one study, Thioflavin T fluorometry was used to determine the effectsof the compounds, and of EDTA (as a negative control). In this assayThioflavin T binds specifically to fibrillar amyloid, and this bindingproduces a fluorescence enhancement at 485 nm that is directlyproportional to the amount of amyloid fibrils formed. The higher thefluorescence, the greater the amount of amyloid fibrils formed (Naki etal., Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410,1993; Amyloid Int. J. Exp. Clin. Invest 2:1-6, 1995).

In this study, 25 μM of pre-fibrillized Aβ 1-42 (Bachem Inc) wasincubated at 37° C. for 3 days either alone, or in the presence of oneof the compounds or EDTA (at Aβ:test compound weight ratios of 1:1,1:0.1, 1:0.01 or 1:0.001). Following 3-days of co-incubation, 50 μl ofeach incubation mixture was transferred into a 96-well microtiter platecontaining 150 μl of distilled water and 50 μl of a Thioflavin Tsolution (i.e. 500 mM Thioflavin T in 250 mM phosphate buffer, pH 6.8).The fluorescence was read at 485 nm (444 nm excitation wavelength) usingan ELISA plate fluorometer after subtraction with buffer alone orcompound alone, as blank.

The results of the 3-day incubations are presented below. For example,whereas EDTA caused no significant inhibition of Aβ 1-42 fibrils at allconcentrations tested, the compounds all caused a dose-dependentdisruption/disassembly of preformed Aβ1-42 fibrils to some extent. Themost efficacious compounds to disrupt pre-formed Aβ 1-42 fibrilsappeared to be compounds #3, 4, 21, 51, 73 and 78. For example, compound#4 caused a significant (p<0.01) 97.4±0.40% inhibition when used at anAβ:test compound wt/wt ratio of 1:0.1, and a 69.4±1.17% disruption whenused at an Aβ:compound wt/wt ratio of 1:0.01. Under the same conditions(i.e. Aβ:test compound wt/wt ratio of 1:0.1), compound #3 caused an57.8±6.36% disruption, compound #21 caused a 81.0±1.31% disruption,compound #51 caused 94.9±0.24% disruption, compound #73 caused a70.9±3.04% disruption, and compound #78 caused a 89.7±1.8% disruption.This study indicated that the compounds of this invention are potentdisruptors/inhibitors of Alzheimer's disease type Aβ fibrils, andusually exert their effects in a dose-dependent manner.

TABLE 1 Thioflavin T fluorometry data - disruption of Aβ 1-42Alzheimer's fibrils % Inhibition Aβ (result ± S.D.) at Aβ:test compoundwt/wt ratio given Test Compound # 1:1 1:0.1 1:0.01 1:0.001 EDTA(control) 11.3 ± 9.67  0.0 ± 7.12  0.0 ± 4.88 0.0 ± 3.01  1 97.3 ± 0.2364.8 ± 1.98 19.2 ± 4.31 0.0 ± 3.07  3 99.5 ± 0.10 57.8 ± 6.36 53.1 ±1.67 5.5 ± 1.99  4 98.5 ± 0.77 97.4 ± 0.40 69.4 ± 1.17 26.8 ± 4.80   870.8 ± 2.57 65.5 ± 0.17 24.7 ± 3.51 4.9 ± 2.27  9 95.1 ± 0.13 34.9 ±1.69  2.0 ± 10.75 10.6 ± 0.93  12 99.7 ± 0.17 82.0 ± 1.13 10.8 ± 21.90.0 ± 34.9 19 99.1 ± 0.56 91.1 ± 0.66 46.2 ± 2.98 10.8 ± 1.38  21 98.6 ±0.54 81.0 ± 1.31 48.2 ± 8.29 8.9 ± 2.13 23 46.7 ± 4.62 26.2 ± 4.37 16.5± 4.02 0.0 ± 3.72 26 37.8 ± 5.50 11.7 ± 3.67  0.0 ± 2.19 0.0 ± 3.24 5199.4 ± 0.05 94.9 ± 0.24 55.3 ± 5.23 29.0 ± 25.2  52 93.7 ± 0.41 53.6 ±2.42 12.1 ± 0.78 0.0 ± 6.67 57 88.4 ± 2.73 60.2 ± 3.12 19.0 ± 6.33 17.7± 7.43  58 94.8 ± 1.67 76.0 ± 2.57 33.2 ± 5.16 20.5 ± 6.27  61 100.0 ±0.41  80.1 ± 4.76 16.9 ± 1.39 26.0 ± 7.51  63 85.3 ± 0.91  23.6 ± 25.75 57.5 ± 10.64 1.6 ± 9.47 66 100.0 ± 0.68  78.3 ± 4.17 42.0 ± 2.36 27.1 ±3.51  67 98.3 ± 2.19 50.9 ± 8.32  34.0 ± 14.07 13.7 ± 6.05  73 99.4 ±0.42 70.9 ± 3.04  28.7 ± 10.27  0.0 ± 29.43 75 99.0 ± 0.63 84.4 ± 0.9431.6 ± 4.74 17.0 ± 4.20  76 99.3 ± 1.35 86.5 ± 1.18 40.9 ± 3.76 12.2 ±5.98  78  100 ± 0.78 89.7 ± 1.18 57.8 ± 4.63 22.4 ± 5.63 Part B: SDS-PAGE/Western Blot Data

The disruption of Aβ 1-42, even in its monomeric form, was confirmed bya study involving the use of SDS-PAGE and Western blotting methods (notshown). In this latter study, triplicate samples of pre-fibrillized Aβ1-42 (25 μM) was incubated at 37° C. for 3 days, alone or in thepresence of the compounds or EDTA. Five micrograms of each sample wasthen filtered through a 0.2 μm filter. Protein recovered from thefiltrate was then loaded, and ran on a 10-20% Tris-Tricine SDS-PAGE,blotted to nitrocellulose and detected using an Aβ-antibody (clone 6E10;Senetek). In this study, Aβ 1-42 was detected as a ˜4 kilodalton band(i.e. monomeric Aβ) following incubation alone, or in the presence ofEDTA, at 3 days. For example, Aβ 1-42 monomers were not detectedfollowing incubation of Aβ 1-42 with compounds 4, 19, 21, 51, 58, 66,75, 76 and 78 suggesting that these compounds were capable of causing adisappearance of monomeric Aβ 1-42. This study confirmed that thesecompounds are also capable of causing a disruption/removal of monomericAβ 1-42.

Part C: Congo Red Binding Data

In the Congo red binding assay the ability of a test compound to alteramyloid (in this case, Aβ) binding to Congo red is quantified. In thisassay, Aβ 1-42 and test compounds were incubated for 3 days and thenvacuum filtered through a 0.2 μm filter. The amount of Aβ 1-42 retainedin the filter was then quantitated following staining of the filter withCongo red. After appropriate washing of the filter, any lowering of theCongo red color on the filter in the presence of the test compound(compared to the Congo red staining of the amyloid protein in theabsence of the test compound) was indicative of the test compound'sability to diminish/alter the amount of aggregated and congophilic Aβ.

In one study, the ability of Aβ fibrils to bind Congo red in the absenceor presence of increasing amounts of the compounds or EDTA (at Aβ:testcompound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001) was determined.The results of 3-day incubations are presented in Table 2 below. WhereasEDTA caused no significant inhibition of Aβ1-42 fibril binding to Congored at all concentrations tested, the compounds caused a dose-dependentinhibition of Aβ binding to Congo red. For example, compound #4 caused asignificant (p<0.01) 73.0±0.90% inhibition of Congo red binding to Aβ1-42 fibrils when used at an Aβ:test compound wt/wt ratio of 1:1, and asignificant (p<0.01) 46.8±1.28% inhibition of Congo red binding whenused at an Aβ:test compound wt/wt ratio of 1:0.1, and a significant(p<0.01) 16.4±2.02% inhibition of Congo red binding when used at anAβ:test compound wt/wt ratio of 1:0.01. In another example, syntheticanalog compound #3 caused a significant (p<0.01) 91.6±5.19% inhibitionof Congo red binding to Aβ 1-42 fibrils when used at an Aβ:test compoundwt/wt ratio of 1:1, and a significant (p<0.01) 35.7±3.29% inhibition ofCongo red binding when used at an Aβ:test compound wt/wt ratio of1:0.01. This study also indicated that compounds of this invention arepotent inhibitors of Aβ fibril binding to Congo red, and usually exerttheir effects in a dose-dependent manner.

TABLE 2 Congo red binding data % Inhibition Aβ (result ± S.D.) atAβ:test compound wt/wt ratio given Test Compound # 1:1 1:0.1 1:0.011:0.001 EDTA (control)  1.1 ± 7.02  3.6 ± 8.68 0.0 ± 3.91 7.91 ± 3.61  1 42.4 ± 1.58  8.0 ± 1.80 3.9 ± 0.66 0.0 ± 3.54  3 91.6 ± 5.19 35.7 ±3.29 7.4 ± 1.51 1.7 ± 4.21  4 73.0 ± 0.90 46.8 ± 1.28 16.4 ± 2.02  2.3 ±1.80  8 17.7 ± 1.86  9.7 ± 0.69 1.1 ± 0.96 0.0 ± 3.55  9 46.8 ± 1.5010.9 ± 2.18 0.0 ± 2.15 3.1 ± 3.66 12 63.0 ± 1.63 20.8 ± 2.22 17.9 ±7.33  4.1 ± 6.60 19 48.1 ± 2.00 22.4 ± 2.19 7.4 ± 2.20 0.0 ± 1.01 2166.2 ± 1.26 33.9 ± 1.02 9.3 ± 5.68 3.6 ± 0.58 23 10.7 ± 2.84  2.9 ± 0.430.0 ± 0.72 12.3 ± 6.57  26  4.5 ± 2.03  0.0 ± 1.35 6.1 ± 4.26 0.0 ± 2.6451 78.6 ± 1.49 46.7 ± 1.29 20.5 ± 11.48  6.0 ± 11.47 52 35.4 ± 1.28 12.7± 2.35 0.0 ± 1.29 0.0 ± 3.68 57 44.8 ± 0.77 14.2 ± 1.56 0.1 ± 2.09 0.0 ±4.73 58 52.2 ± 2.65 21.1 ± 3.67 6.6 ± 3.49 2.5 ± 4.22 61 48.9 ± 4.69 24.6 ± 10.85 2.0 ± 2.89 0.0 ± 4.06 63 32.5 ± 5.66  8.5 ± 8.01 20.1 ±10.35 0.0 ± 1.93 66 55.9 ± 6.83  27.7 ± 11.26 7.7 ± 0.19 0.6 ± 6.61 67 31.5 ± 11.25  13.8 ± 11.25 8.2 ± 7.08 0.0 ± 4.98 73 53.4 ± 1.84 22.6 ±3.51 0.6 ± 5.04  0.0 ± 15.17 75 59.2 ± 3.23 12.8 ± 0.59 6.8 ± 2.55 2.4 ±2.95 76 66.6 ± 0.68 27.8 ± 7.71 4.1 ± 2.23 0.3 ± 5.1  78 71.1 ± 1.0939.9 ± 3.94 15.4 ± 1.39  3.5 ± 1.33Part D—Circular Dichroism Spectroscopy Data

Circular dichroism (CD) spectroscopy is a method that can be used todetermine the effects of test compounds to disrupt the secondarystructure conformation of amyloid fibrils. In one study, as described inthis example, circular dichroism spectroscopy was used to determine theeffects of different compounds of the invention on β-sheet conformationof Aβ 1-42 fibrils. For this study, Aβ 1-42 (Bachem Inc., Torrance,Calif.) was first dissolved in a 2 mM NaOH solution, maintaining the pHof these solutions above 10. Aβ 1-42 peptides (at 25 μM), in the absenceor presence of test compounds, were made up in 150 nM NaF, 50 mMphosphate buffer, pH 7.4 with 10% trifluoroethanol. Aβ 1-42 was thenincubated at 37° C. in the absence or presence of different compounds atan Aβ:test compound wt/wt ratios of 1:0.1, 1:1 and 1:10. After 3 days ofincubation, CD spectra were recorded on a Jasco 810 spectropolarimeter(Easton, Md.). All CD spectra were collected with 0.05 cm quartz cells.Wavelength traces were scanned from 190-260 nm at 0.5 nm increments witha bandwidth of 5 nm, at a scan speed of 10 nm/minute, a response time of32 seconds, and a data pitch of 0.5 nm. The whole system wasequilibrated and continuously flushed with nitrogen at 10 ml/minute. Fordata processing, the average of 5 replicates of “test-compound” spectrawere subtracted from the average of 5 replicates of “Aβ 1-42+testcompound” spectra to determine the effects of each test compound ondisruption of Aβ 1-42 fibrils. Ellipticity in degrees was converted toMRE ([Q]; molar residue ellipticity) using the formula[Q]=100·Q·RMW/d·c; where Q is the ellipticity in degrees; RMW is theaverage residue molecular weight (˜107 daltons for Aβ 1-42); d is thepathlength in cm (i.e. 0.05 cm); and c is the concentration in mg/ml(i.e. 0.1 mg/ml).

FIG. 1 shows some of the CD spectra generated in this study. Aβ 1-42alone in 10% TFE PBS buffer usually demonstrated the typical CD spectraof an amyloid protein with significant β-sheet structure, asdemonstrated by the minima observed at 218 nm. However, in the presenceof test compounds (such as the compounds #4, 12, 51 and 61 shown inFIG. 1) a marked disruption of β-sheet structure in Aβ 1-42 fibrils wasevident (with a significant increase in random coil or α-helix) as shownby the flattening out of the minima observed at 218 nm (compare to Aβ1-42 alone). This was usually observed at both 3 days (as seen inFIGS. 1) and 7 days (not shown) following co-incubation of Aβ 1-42fibrils with the compounds.

FIG. 2 shows the effect of compound #78 on disruption of Aβ 1-42fibrils. As shown in this figure, Aβ 1-42 alone demonstrates the typicalCD spectra of a predominant β-sheet structure, with a marked minimaobserved at 218 nm. However, in the presence of compound #78 at 3 days,there is a marked decrease in the minima usually observed at 218 nm(with Aβ 1-42 only), indicative of a disruption of the β-sheet structureof Aβ 1-42 fibrils.

FIG. 3 shows the dose-response effects of compounds #12, 51 and 61 ondisruption of the β-sheet structure of Aβ 1-42 fibrils. As an example,increasing concentrations of test compounds #12, 51 and 61 (at Aβ:testcompounds wt/wt ratios of 1:0.1, 1:1 and 1:10) caused a generaldisruption of β-sheet structure as demonstrated by the dose-dependentdecrease in the minima observed at 218 nm (when compared to the minimaat 218 nm observed with Aβ 1-42 only). Compound #51 was particularlyeffective when used at an Aβ:test compound wt/wt ratio of 1:10 and wasshown to completely disrupt the β-sheet structure of Aβ 1-42 fibrils asshown by the complete flattening out of the minima observed at 218 nm(compare to Aβ 1-42 alone) (FIG. 3).

The CD studies demonstrate that the compounds of this invention have theability to disrupt/disassemble the β-sheet structure characteristic ofAlzheimer's Aβ fibrils. The results of the studies also confirm theprevious examples using Thioflavin T fluorometry, SDS-PAGE/ECL, andCongo red binding type assays, that the compounds of this invention arepotent anti-amyloid agents.

Example 26 Compounds of This Invention are Potent Disrupters of Type 2Diabetes IAPP Fibrils

The compounds prepared in the synthetic Examples were found also to bepotent disruptors/inhibitors of type 2 diabetes IAPP fibrils. In a setof studies, the efficacy of the compounds to cause adisassembly/disruption of pre-formed IAPP fibrils of type 2 diabetes wasanalyzed.

Part A—Thioflavin T Fluorometry Data

In one study, Thioflavin T fluorometry was used to determine the effectsof the compounds, and of EDTA (as a negative control). In this assayThioflavin T binds specifically to fibrillar amyloid, and this bindingproduces a fluorescence enhancement at 485 nm that is directlyproportional to the amount of IAPP fibrils present. The higher thefluorescence, the greater the amount of IAPP fibrils present (Naki etal, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410,1993; Amyloid. Int. J. Exp. Clin. Invest. 2:1-6, 1995).

In this study, 25 μM of pre-fibrillized IAPP (Bachem Inc) was incubatedat 37° C. for 3 days either alone, or in the presence of one of thecompounds or EDTA (at IAPP:test compound weight ratios of 1:1, 1:0.1,1:0.01 or 1:0.001). Following 3-days of co-incubation, 50 μl of eachincubation mixture was transferred into a 96-well microtiter platecontaining 150 μl of distilled water and 50 μl of a Thioflavin Tsolution (i.e. 500 mM Thioflavin Tin 250 mM phosphate buffer, pH 6.8).The fluorescence was read at 485 nm (444 nm excitation wavelength) usingan ELISA plate fluorometer after subtraction with buffer alone orcompound alone, as blank

The results are presented in Table 3 below. For example, whereas EDTAcaused no significant inhibition of IAPP fibrils at all concentrationstested, the compounds all caused a dose-dependent disruption/disassemblyof pre-formed IAPP fibrils to various extents. The most efficaciouscompounds to disrupt IAPP fibrils appeared to be compounds #3, 4, 23,63, and 78. For example, compound #3 caused a significant (p<101)97.7±0.19% inhibition when used at an IAPP:test compound ratio of 1:0.1,and a 79.9±1.47% disruption when used at a IAPP:compound wt/wt ratio of1:0.01. Under the same conditions (i.e. IAPP:test compound wt/wt ratioof 1:0.1), compound #4 caused a 96.0±1.0% disruption, compound #23caused a 67.2±18.35% disruption, compound #63 caused a 84.2 ±1.16%disruption, compound #78 caused 92.4±0.27% disruption, and compound #26caused a 45.9±17.73% disruption. This study indicated that the compoundsof this invention are also potent disruptors/inhibitors of type 2diabetes IAPP fibrils, and usually exert their effects in adose-dependent manner.

TABLE 3 Thioflavin T fluorometry data - disruption of type 2 diabetesIAPP fibrils % Inhibition IAPP (result ± S.D.) at IAPP:test compoundwt/wt ratio given Test Compound # 1:1 1:0.1 1:0.01 1:0.001 EDTA(control)  4.4 ± 9.23  0.1 ± 2.59  0.0 ± 5.23 4.2 ± 1.05  1 99.0 ± 0.1193.0 ± 1.27 57.3 ± 0.16 6.4 ± 4.40  3  100 ± 0.20 97.7 ± 0.19 79.9 ±1.47 30.7 ± 6.71   4 99.7 ± 0.23 96.0 ± 0.10 63.2 ± 2.09 17.3 ± 4.07   872.8 ± 1.77 67.8 ± 1.74 29.6 ± 5.97 11.4 ± 12.78 12 99.9 ± 0.19 86.0 ±0.76 37.5 ± 0.76 13.0 ± 10.34 19 100.0 ± 0.24  94.0 ± 0.10 51.7 ± 2.9816.7 ± 10.20 21 98.5 ± 0.06 85.4 ± 0.86 25.8 ± 3.61  5.4 ± 15.41 23 85.2± 0.55  67.2 ± 18.35  44.3 ± 32.47 27.3 ± 45.38 26 52.5 ± 2.44  45.9 ±17.73 24.6 ± 6.77 3.7 ± 4.67 51 99.9 ± 0.11 96.6 ± 1.00 56.6 ± 1.69 11.8± 6.45  52 97.9 ± 0.19 86.9 ± 3.09 49.2 ± 4.47 16.0 ± 8.42  57 94.1 ±0.46 73.2 ± 1.19 37.3 ± 0.78 1.9 ± 5.24 58 98.1 ± 1.04 87.6 ± 1.16 48.8± 2.05 8.9 ± 6.87 61 96.8 ± 0.47 83.6 ± 1.27 35.4 ± 5.68 0.5 ± 6.33 6394.9 ± 0.65 84.2 ± 1.16 56.2 ± 8.77 19.0 ± 0.30  66 98.5 ± 0.06 94.0 ±2.88 47.6 ± 8.16 11.1 ± 5.28  67 98.6 ± 0.22 81.4 ± 6.96 34.8 ± 1.8716.1 ± 12.40 75  100 ± 0.35 90.0 ± 0.27 43.9 ± 5.34 6.0 ± 6.46 76 99.6 ±1.01 87.5 ± 1.89 41.5 ± 6.67 9.0 ± 0.32 78 99.5 ± 0.26 92.4 ± 0.27 58.3± 1.20 15.3 ± 4.73 Part B: Congo Red Binding Data

In the Congo red binding assay the ability of a given test compound toafter amyloid (in this case, IAPP) binding to Congo red is quantified.In this assay, IAPP and test compounds were incubated for 3 days andthen vacuum filtered through a 0.2 μm filter. The amount of IAPPretained in the filter was then quantitated following staining of thefilter with Congo red. After appropriate washing of the filter, anylowering of the Congo red color on the filter in the presence of thetest compound (compared to the Congo red staining of the amyloid proteinin the absence of the test compound) was indicative of the testcompound's ability to diminish/alter the amount of aggregated andcongophilic IAPP.

In the study, the ability of IAPP fibrils to bind Congo red in theabsence or presence of increasing amounts of the compounds or EDTA (atIAPP:test compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001) wasdetermined. The results of 3-day incubations are presented in Table 4below. Whereas EDTA caused no significant inhibition of IAPP fibrilbinding to Congo red at all concentrations tested, the compounds usuallycaused a dose-dependent inhibition of IAPP binding to Congo red. Forexample, compound #3 caused a significant (p<0.01) 55.5±2.68% inhibitionof Congo red binding to IAPP fibrils when used at an IAPP:test compoundwt/wt ratio of 1:1, and a significant (p<0.01) 37.9 ±3.10% inhibition ofCongo red binding when used at an IAPP:test compound wt/wt ratio of1:0.1. Compound #4 caused a significant (p<0.01) 68.9±1.22% inhibitionof Congo red binding to IAPP fibrils when used at an IAPP:test compoundwt/wt ratio of 1:1, and a 25.4±4.68% inhibition of Congo red bindingwhen used at a NAC-test compound wt/wt ratio of 1:0.01. This studyindicated that compounds of this invention are also potent inhibitors oftype 2 diabetes IAPP fibril binding to Congo red, and usually exerttheir effects in a dose-dependent manner.

TABLE 4 Congo red binding data % Inhibition IAPP (result ± S.D.) atIAPP:test compound wt/wt ratio given Test Compound # 1:1 1:0.1 1:0.011:0.001 EDTA  0.0 ± 3.69  0.0 ± 1.91  3.6 ± 2.83 6.6 ± 2.27 (control)  140.7 ± 2.49 10.6 ± 3.40 18.6 ± 4.05 6.4 ± 2.07  3 55.5 ± 2.68 37.9 ±3.10 16.3 ± 1.13 11.1 ± 5.26   4 68.9 ± 1.22 25.4 ± 4.68  9.0 ± 0.51 0.0± 1.05  8  0.0 ± 2.84  0.0 ± 2.94  7.2 ± 2.27 0.0 ± 6.46 12 39.8 ± 0.26 8.3 ± 0.85  6.9 ± 2.45 0.0 ± 2.40 19 49.3 ± 3.97 21.0 ± 3.70  6.0 ±0.78 2.9 ± 4.40 21 35.9 ± 0.21 10.4 ± 3.53  5.1 ± 4.53 0.0 ± 2.10 23 5.5 ± 2.33  4.5 ± 4.12  9.3 ± 1.40 5.1 ± 2.45 26  0.0 ± 1.21  7.5 ±2.83  5.3 ± 6.14 10.8 ± 2.63  51 55.6 ± 1.48 27.5 ± 3.49  3.6 ± 2.59 1.6± 1.01 52 31.3 ± 0.27 11.5 ± 1.21 11.0 ± 3.27 10.2 ± 0.52  57 15.7 ±3.77  8.9 ± 3.90  8.5 ± 3.19 4.5 ± 0.64 58 24.5 ± 0.57  0.7 ± 6.21  4.6± 2.35 0.0 ± 1.93 61 23.7 ± 0.39  0.0 ± 7.07  4.0 ± 1.78 0.0 ± 3.87 6315.4 ± 1.34  4.5 ± 1.62 11.7 ± 2.26 0.0 ± 2.25 66 41.4 ± 3.84 15.7 ±2.53  5.7 ± 4.23 4.8 ± 1.86 67 26.3 ± 2.76  5.5 ± 2.52 10.6 ± 1.29 0.0 ±3.45 75 49.0 ± 1.17  7.4 ± 0.70 11.3 ± 2.24 2.9 ± 0.69 76 53.9 ± 5.4416.5 ± 2.60 14.2 ± 2.25 3.4 ± 1.07 78 56.3 ± 5.32 16.7 ± 6.80 19.9 ±2.12 6.6 ± 3.04

Example 27 Compounds of This Invention are Potent Disrupters ofParkinson's Disease NAC Fibrils

The tested compounds of this invention were found also to be potentdisruptors/inhibitors of Parkinson's disease NAC fibrils. NAC is a35-amino acid fragment of α-synuclein that has been demonstrated to formamyloid-like fibrils when incubated at 37° C. for a few days. It is theamyloidogenic fragment of α-synuclein and is postulated to play animportant role in the pathogenesis of Parkinson's disease and othersynucleinopathies. In a set of studies, the efficacy of the compounds tocause a disassembly/disruption of pre-formed NAC fibrils of Parkinson'sdisease was analyzed.

Part A—Thioflavin T Fluorometry Data

In one study, Thioflavin T fluorometry was used to determine the effectsof compounds #1, 3, 23, 26, 52, 63, 66, 67, and EDTA (as a negativecontrol). In this assay, Thioflavin T binds specifically to NAC fibrils,and this binding produces a fluorescence enhancement at 485 nm that isdirectly proportional to the amount of NAC fibrils present. The higherthe fluorescence, the greater the amount of NAC fibrils present (Naki etal, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410,1993; Amyloid. Int J. Exp. Clin. Invest. 2:1-6, 1995).

In this study, 25 μM of pre-fibrillized NAC (Bachem Inc) was incubatedat 37° C. for 3 days either alone or in the presence of dihydroxysynthetic analog compounds #1, 3, 23, 26, 52, 63, 66, 67, or EDTA (atNAC:test compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001).Following 3-days of co-incubation, 50 μl of each incubation mixture wastransferred into a 96-well microtiter plate containing 150 μl ofdistilled water and 50 μl of a Thioflavin T solution (i.e. 500 mMThioflavin T in 250 mM phosphate buffer, pH 6.8). The fluorescence wasread at 485 nm (444 nm excitation wavelength) using an ELISA platefluorometer after subtraction with buffer alone or compound alone, asblank.

The results of the 3-day incubations are presented below in Table 5. Forexample, whereas EDTA caused no significant inhibition of NAC fibrils atall concentrations tested, compounds 1, 3, 52, 63, 66, and 67 all causeda dose-dependent disruption/disassembly of pre-formed NAC fibrils tovarious extents. For example, compound #3 caused a significant (p<0.01)91.0±1.99% inhibition when used at an NAC:test compound ratio of 1:0.1,and a 93.9±0.77% disruption when used at a NAC:compound wt/wt ratio of1:0.01. Under the same conditions (i.e. NAC:test compound wt/wt ratio of1:0.1), compound #1 caused a 99.5±0.53% disruption, compound #26 causeda 61.3±6.52% disruption, compound #52 caused a 89.2±1.49% disruption,compound #66 caused a 82.5±5.37% disruption, and compound #67 caused a50.0±7.03% disruption. This study indicated that compounds of thisinvention are potent disruptors/inhibitors of Parkinson's disease NACfibrils, and usually exert their effects in a dose-dependent manner.

TABLE 5 Thioflavin T fluorometry data - disruption of Parkinson'sdisease NAC fibrils % Inhibition NAC (result ± S.D.) at NAC:testcompound wt/wt ratio given Test Compound # 1:1 1:0.1 1:0.01 1:0.001 EDTA(control) 20.0 ± 11.8  0.0 ± 5.87  0.0 ± 10.87 0.0 ± 11.6  1 100.0 ±1.00  99.5 ± 0.53 68.2 ± 2.55 0.0 ± 7.14  3 98.0 ± 1.78 91.0 ± 1.99 93.9± 0.77 67.3 ± 6.37  23 58.0 ± 8.43  53.3 ± 12.02 35.6 ± 9.73  0.0 ±26.42 26 70.4 ± 3.22 61.3 ± 6.52 56.8 ± 4.60  0.0 ± 16.88 52 99.7 ± 1.9389.2 ± 1.49 79.6 ± 6.43 13.8 ± 10.49 63  45.6 ± 31.03  34.5 ± 17.15 33.0± 1.69 17.3 ± 12.57 66 98.9 ± 0.65 82.5 ± 5.37 43.4 ± 3.45 30.5 ± 9.55 67 97.4 ± 1.19 50.0 ± 7.03 30.6 ± 5.75 11.9 ± 15.98Part B: Congo Red Binding Data

In the Congo red binding assay, the ability of a given test compound toalter amyloid (in this case, NAC) binding to Congo red is quantified. Inthis assay, NAC and test compounds were incubated for 3 days and thenvacuum filtered through a 0.2 μm filter. The amount of NAC retained inthe filter was then quantitated following staining of the filter withCongo red. After appropriate washing of the filter, any lowering of theCongo red color on the filter in the presence of the test compound(compared to the Congo red staining of the amyloid protein in theabsence of the test compound) was indicative of the test compound'sability to diminish/alter the amount of aggregated and congophilic NAC.

In one study, the ability of NAC fibrils to bind Congo red in theabsence or presence of increasing amounts of compounds #1, 3, 23, 26,63, 66, 67, or EDTA (at NAC:test compound weight ratios of 1:1, 1:0.1,1:0.01 or 1:0.001) was determined. The results of 3-day incubations arepresented in Table 6, Whereas EDTA caused no significant inhibition ofNAC fibril binding to Congo red at all concentrations tested, thecompounds tested caused a dose-dependent inhibition of NAC binding toCongo red as demonstrated in Table 6 below. For example, compound #3caused a significant (p<0.01) 94.4±2.48% inhibition of Congo red bindingto NAC fibrils when used at a NAC:test compound wt/wt ratio of 1:1, anda significant (p<0.01) 83.2±3.57% inhibition of Congo red binding whenused at a NAC:test compound wt/wt ratio of 1:0.1. In comparison,compound #1 caused a 75.4±2.96% inhibition of Congo red binding to NACfibrils when used at a NAC:test compound wt/wt ratio of 1:1, and an75.9±2.48% inhibition of Congo red binding when used at a NAC:testcompound wt/wt ratio of 1:0.1. In another example, synthetic analogcompound #67 caused a significant (p<0.01) 81.2+/−2.87% inhibition ofCongo red binding to NAC fibrils when used at an NAC:test compound wt/wtratio of 1:1, and a significant (p<0.01) 47.7±8.20% inhibition of Congored binding when used at a NAC:test compound wt/wt ratio of 1:0.01. Inanother example, compound #26 caused a significant 34.4±10.19%inhibition of Congo red binding when used at a NAC:test compound ratioof 1:1, and a 36.7%±5.59% inhibition of Congo red binding when used at aNAC:test compound ratio of 1:0.1. This study also indicated thatcompounds of this invention are also potent inhibitors of Parkinson'sdisease type NAC fibril binding to Congo red, and usually exert theireffects in a dose-dependent manner.

TABLE 6 Congo red binding data - disruption of Parkinson's disease NACfibrils % Inhibition NAC (result ± S.D.) at NAC:test compound wt/wtratio given Test Compound # 1:1 1:0.1 1:0.01 1:0.001 EDTA (control)  0.2± 7.33  0.0 ± 38.26  0.0 ± 22.0 0.0 ± 20.57  1 75.4 ± 2.96 75.9 ± 2.5840.7 ± 4.23 0.0 ± 11.39  3 94.4 ± 2.48 83.2 ± 3.57 81.7 ± 2.82 65.2 ±5.40  23 41.0 ± 8.54  30.3 ± 12.06 25.6 ± 5.37 0.0 ± 9.00  26  34.4 ±10.19 36.7 ± 5.59 36.4 ± 0.67 0.0 ± 27.34 52 73.8 ± 3.15 71.2 ± 7.1778.9 ± 4.76 0.0 ± 24.43 63 54.5 ± 7.56  9.3 ± 10.5 34.0 ± 3.66 0.0 ±30.84 66 81.1 ± 1.74 72.4 ± 1.79 51.0 ± 9.50 19.5 ± 37.59  67 81.2 ±2.87 47.7 ± 8.20  39.2 ± 10.25 15.5 ± 41.42 

Example 28 Other bis- and tris-dihydroxyaryl Compounds of the Invention

Besides the 24 compounds described in detail in. Examples 1-24, thisExample describes other bis- and tris(dihydroxyaryl) compounds that alsoserve as potent disruptor/inhibitors of amyloid fibrils in Alzheimer'sdisease (i.e. Aβ), type 2 diabetes (i.e. IAPP), other amyloid diseases,as well as in Parkinson's disease (i.e. α-synuclein/NAC) and othersynuclein fibril diseases. A common structural motif that is present inall of the compounds disclosed herein is the presence of two or threedihydroxyaryl groups. These compounds are compounds #2, 5, 6, 7, 10, 11,13, 14, 15, 16, 17, 18, 20, 22, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 54,55, 56, 59, 60, 62, 64, 65, 68, 69, 70, 71, 72, 74, 79 and 80. These arealso referred respectively to as DC-0002, DC-0005, DC-0006, DC-0007,DC-0010, DC-0011, DC-0013, DC-0014, DC-0015, D00016, DC-0017, DC-0018,DC-0020, DC-0022, DC-0024, DC-0025, DC-0027, DC-0028, DC-0029, DC-0030,DC-0031, DC-0032, DC-0033, DC-0034, DC-0035, DC-0036, DC-0037, DC-0038,DC-0039, DC-0040, DC-0041, DC-0042, DC-0043, DC-0044, DC-0045, DC-0046,DC-0047, DC-0048, DC-0049, DC-0050, DC-0053, DC-0054, DC-055, DC-0056,DC-0059, DC-0060, DC-0062, DC-0064, DC-0065, DC-0068, DC-0069, DC-0070,DC-0071, DC-0072, DC-0074, DC-0079 and DC-0080, respectively. [Compound#77 also appears in the compound chart following].

These compounds may be prepared by the methods used to produce thecompounds illustrated in Examples 1 through 23 and variations thereofeasily determinable by a person of ordinary skill in the art. Thus, forexample, compounds 10 and 11 may be prepared by the method used forcompound 9, substituting N,N′-dimethylethylenediamine and2,5-diaza[2.2.1]bicycloheptane for the piperazine of Example 5,compounds #17 and 18 may be prepared by the method used for compound 19,substituting cyclohexanone and N-methyl-4-piperidinone for the tropinoneof Example 7; compounds 24 and 25 may be prepared by the method used forcompound 12, substituting N,N′-dimethylethylenediamine and2,5-diaza[2.2.1]bicycloheptane for the trans-1,2-diaminocyclohexane ofExample 6, and so on. A person of ordinary skill in the art will have nodifficulty, having regard to that skill and this disclosure, inpreparing the compounds illustrated above or the compounds of theformula given in claim 1.

Example 29 Compound of the Invention with Rigid Scaffolds

This Example illustrates six further compounds of this invention(compounds #81, 82, 83, 84, 85, and 86 or DC-0081 through DC-0086) thatalso serve as potent disruptor/inhibitors of amyloid fibrils inAlzheimer's disease (i.e. Aβ), type 2 diabetes (i.e. IAPP), otheramyloid diseases, as well as in Parkinson's disease (i.e.α-synuclein/NAC) and other synuclein fibril diseases. These compoundshave relatively rigid scaffold structures. The synthesis of compound 85is given in Example 24.

Example 30 Methylenedioxy Analogs

A strategy for the delivery of the dihydroxyaryl compounds of thisinvention to improve and/or cause more favorable metabolism andbioavailability characteristics involves the protection of the hydroxygroups of the dihydroxyaryl compounds with methylenedioxy groups. Thisstrategy is exemplified in the 80 structures shown below, and is equallyapplicable to protect the dihydroxyaryl groups of compounds #81-86.Methylenedioxy analogs represent intermediate hydroxy protectingstructures that are made to successfully complete the synthesis of thedihydroxyaryl compounds described in the invention. These closed-ringcompounds also tend to be more stable, and hydrophobic (waterinsoluble), and less likely to be altered or degraded due to theoxidation that could occur if hydroxyl groups were present. In addition,these compounds make good prodrugs especially for delivery to the braindue to their hydrophobic nature. Hydrophobic compounds that are lipidsoluble tend to be attractive compounds for brain delivery since theyare usually able to penetrate the blood-brain-barrier.

The methylenedioxy analogs are generally available as intermediates inthe synthesis of the corresponding dihydroxyaryl compounds, as may beseen from the syntheses illustrated in Examples 1-23. These compoundsare expected to be efficacious in their ability to cause adisruption/disassembly and inhibition of amyloid and synuclein fibrils,once the methylenedioxy structures are cleaved to yield hydroxyl groups.Conversion of the hydroxyl groups to methylenedioxy derivatives alsoyields prodrugs that are believed to improve toxicity (i.e. being lesstoxic), metabolism (since the OH groups will be less likely to bealtered by methylation, glucuronidation and sulfation), andbioavailability. In this prodrug concept, it is believed that theprodrug conversion takes place in the plasma (following its protectionthrough the gut), and closer to its appropriate target tissue (systemicorgans and/or brain). Enzymes in the blood and appropriate tissues arebelieved to be able to cleave the methylenedioxy group on these analogsto yield the dihydroxy structures to achieve the observed efficacyagainst the diseases described earlier in the application such asAlzheimer's disease, type 2 diabetes, Parkinson's disease and otheramyloid diseases and synucleinopathies.

Example 31 Acylated Compounds

Another potential strategy for the delivery of the bis- andtris-dihydroxyaryl compounds of this invention to improve and/or causemore favorable metabolism and bioavailability characteristics, involvesmethods of protecting the hydroxy groups as their pharmaceuticallyacceptable esters. Ester groups replacing the hydroxy groups also tendto make the compounds more stable, and less likely to be altered ordegraded due to oxidation of the hydroxyl groups.

The compound table below illustrates the acetyl esters of the 80dihydroxyaryl compounds of Examples 1 -23 and 28 are presented below inwhich the OH groups are replaced by acetyl groups. The illustration ofacetyl esters here is merely exemplary for the class of pharmaceuticallyacceptable esters that are part of the compounds of this invention andmay be prepared by analogous methods. The compounds of Example 29 alsoform pharmaceutically acceptable esters in the same manner, and thesecompounds, though not illustrated in the compound table below, are alsocompounds of this invention.

These compounds are expected to be efficacious in their ability to treatamyloid diseases and synucleinopathies once the ester linkages arecleaved (by enzymes in the plasma or in the brain tissue), and thehydroxyl groups are regenerated. Replacement of the hydroxyl groups withester groups will yield prodrugs that are believed to improve toxicity(i.e. being less toxic), metabolism (since the OH groups will be lesslikely to be altered by methylation, glucuronidation and sulfation), andbioavailability. In this prodrug concept, it is believed that theprodrug conversion takes place in the plasma (following its protectionthrough the gut), and closer to its appropriate target tissue (systemicorgans for the treatment of systemic amyloid diseases and/or brain forthe treatment of Alzheimer's, Parkinson's, type 2 diabetes, and otherAβ, amyloid and synuclein diseases). Enzymes in the blood andappropriate tissues are believed to be able to cleave the ester linkageson these pharmaceutically acceptable esters to yield the dihydroxystructures important for the observed efficacy against Alzheimer'sdisease, other amyloid diseases (such as IAPP fibrils in type 2diabetes), and α-synuclein/NAC fibrils, such as in Parkinson's disease,and other synucleinopathies.

The pharmaceutically acceptable esters of compounds #1 through #86 areprepared by methods well known to persons of ordinary skill in the art,such as by reaction of the dihydroxyaryl compounds with pharmaceuticallyacceptable acids, especially in activated form (such as the acylhalides) and/or in the presence of reagents facilitating esterification(such as an acidic catalyst) and/or under conditions favoringesterification (such as by conducting the reaction under conditionswhere the water formed in the esterification is removed, e.g. bydistillation). Methods of esterification of phenolic hydroxyl groups arewell known to persons of ordinary skill in the art.

Suitable acids for the formation of pharmaceutically acceptable estersare the C₂₋₆ alkanoic acids (acetic acid, propionic acid, and the like),benzoic acid, arytalkanoic acids (phenylacetic acid, and the like);though many other acids are suitable for the formulation ofpharmaceutically acceptable esters, and a person of ordinary skill inthe art will have no difficulty in choosing a suitable acid.

Example 32 Compositions of Compounds of this Invention

The compounds of this invention, as mentioned previously, are desirablyadministered in the form of pharmaceutical compositions. Suitablepharmaceutical compositions, and the method of preparing them, arewell-known to persons of ordinary skill in the art and are described insuch treatises as Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins,Philadelphia, Pa.

Representative compositions are as follows:

Oral Tablet Formulation

An oral tablet formulation of a compound of this invention is preparedas follows:

% w/w Compound of this invention 10.0 Magnesium stearate 0.5 Starch 2.0Hydroxypropylmethylcellulose 1.0 Microcrystalline cellulose 86.5

The ingredients are mixed to homogeneity, then granulated with the aidof water, and the granulates dried. The granulate is then compressedinto tablets sized to give a suitable dose of the compound. The tabletis optionally coated by applying a suspension of a film forming agent(e.g. hydroxypropylmethylcellulose), pigment (e.g. titanium dioxide),and plasticizer (e.g. diethyl phthalate), and drying the film byevaporation of the solvent. The film coat may comprise, for example,2-6% of the tablet weight.

Oral Capsule Formulation

The granulate from the previous section of this Example is filled intohaul gelatin capsules of a size suitable to the intended dose. Thecapsule is banded for sealing, if desired.

Softgel Formulation

A softgel formulation is prepared as follows:

% w/w Compound of this invention 20.0 Polyethylene glycol 400 80.0

The compound is dissolved or dispersed in the polyethylene glycol, and athickening agent added if required. A quantity of the formulationsufficient to provide the desired dose of the compound is then filledinto softgels.

Parenteral Formulation

A parenteral formulation is prepared as follows:

% w/w Compound of this invention 1.0 Normal saline 99.0

The compound is dissolved in the saline, and the resulting solution issterilized and filled into vials, ampoules, and prefilled syringes, asappropriate.

Controlled-Release Oral Formulation

A sustained release formulation may be prepared by the method of U.S.Pat. No. 4,710,384, as follows:

One Kg of a compound of this invention is coated in a modified Uni-Glattpowder coater with Dow Type 10 ethyl cellulose. The spraying solution isan 8% solution of the ethyl cellulose in 90% acetone to 10% ethanol.Castor oil is added as plasticizer in an amount equal to 20% of theethyl cellulose present. The spraying conditions are as follows: 1)speed, 1 liter/hour; 2) flap, 10-15%; 3) inlet temperature, 50° C., 4)outlet temperature, 30° C., 5) percent of coating, 17%. The coatedcompound is sieved to particle sizes between 74 and 210 microns.Attention is paid to ensure a good mix of particles of different sizeswithin that range. Four hundred mg of the coated particles are mixedwith 100 mg of starch and the mixture is compressed in a hand press to1.5 tons to produce a 500 mg controlled release tablet.

Example 33 Compounds of This Invention are Potent Disruptors/Inhibitorsof α-synuclein Fibrils Associated with Parkinson's Disease

Parkinson's Disease is characterized by the accumulation of insolubleintraneuronal aggregates called Lewy Bodies, a major component of whichis α-synuclein (reviewed in Dauer et al., Neuron, 39:889-909, 2003).Since autosomal dominant mutations in α-synuclein cause a subset offamilial Parkinson's disease, and since these mutations increase thelikelihood of α-synuclein to aggregate and form Lewy Bodies, aggregatedα-synuclein is proposed to be directly involved in the etiology anddisease progression (Polymeropoulos et al., Science 276:1197-1199, 1997;Papadimitriou et al., Neurology 52:651-654, 1999). Structural studieshave revealed that similar to extracellular amyloid deposits containingthe amyloid β peptide, intracellular Lewy bodies contain a largeproportion of misfolded proteins with a high degree of β-pleated sheetsecondary structure. These studies were conducted to determine theefficacy of the test compounds in the inhibition/disruption ofα-synuclein fibrils associated with Parkinson's disease.

Therefore, to test the therapeutic potential of the compounds, twocell-based assays were utilized. In both assays, rotenone is used toinduce mitochondrial oxidative stress and α-synuclein aggregation. Thefirst assay utilizes the binding of the fluorescent dye thioflavin S tostructures with high β-sheet content including α-synuclein fibrils.Therefore, quantitative assessment of the extent of thioflavinS-positive staining of fixed cells is used to test the ability of thecompounds to decrease the amount of α-synuclein aggregates. In thesecond assay, cell viability is assessed using the XTT Cytotoxicityassay, which is dependent on intact, functional mitochondria in livecells. Thus, the XTT Cytotoxicity assay is used to test the ability ofthe compounds to ameliorate the mitochondrial toxicity and resultingloss of viability associated with the accumulation of α-synucleinaggregates. Phrased another way, the XTT Cytotoxicity assay is used togauge the compounds neuroprotective efficacy. These studies arepresented in the following examples.

To carry out these studies, a cell culture model was used in which humanα-synuclein aggregation is experimentally induced. BE-M17 humanneuroblastoma cells stably transfected with A53T-mutant humanα-synuclein were obtained. Cell culture reagents were obtained fromGibco/Invitrogen, and cells were grown in OPTIMEM supplemented with 10%FBS, Penicillin (100 units/ml)—Streptomycin (100 μg/ml) and 500 μg/mlG418 as previously described (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000).

Thioflavin S is commonly used to detect amyloid-containing structures msitu, including in brain tissue (Vallet et al., Acta Neuropathol.,83:170-178, 1992), and cultured cells (Ostrerova-Golts et al, J.Neurosci., 20:6048-6054, 2000), whereas thioflavin T is often used as anin vitro reagent to analyze the aggregation of soluble amyloid proteinsinto fibrils enriched in β-pleated sheet structures (LeVine III, Prot.Sci., 2:404-410, 1993). Therefore, Thioflavin S histochemistry was usedon cultured cells to detect aggregates containing a high degree ofβ-pleated structures that formed in response to oxidativestress-inducing agents (in this case rotenone) as previously described,with minor modifications (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000). Briefly, for these studies, cells were grown onPoly-D-Lysine coated glass slide chambers at approximately 3×10⁴cells/cm². After 24 hours, cells were treated with 500 nM, 1 μM or 5 μMrotenone (Sigma) or vehicle (0.05% DMSO) as indicated. Immediately afterrotenone (or vehicle) addition, compounds were added at the indicatedconcentration, or cell culture media only (no compound) in the presenceof rotenone was added. Identical treatments were repeated after 48hours. After an additional 48 hours, cells were fixed for 25 minutes in3% paraformaldehyde. After a PBS wash, the cells were incubated with0.015% thioflavin S in 50% ethanol for 25 minutes, washed twice for fourminutes in 50% ethanol and twice for five minutes in deionized water andthen mounted using an aqueous-based mountant designed to protect againstphotobleaching. Aggregates that bind to thioflavin S were detected witha fluorescent microscope using a High Q FITC filter set (480 to 535 nmbandwidth) and a 20× objective lens unless otherwise indicated. Between8 and 16 representative images per condition were selected, imaged andprocessed by an experimenter who was blinded to treatment conditions. Toassess the amount of thioflavin S-positive aggregates, the total areaper field covered by thioflavin S-positive inclusions was determined.For this purpose, background fluorescence that failed to exceed pre-setsize or pixel intensity threshold parameters was eliminated usingQ-capture software. Spurious, non-cell associated fluorescence wasmanually removed. Unless indicated otherwise, data represent groupmeans±SEM. Statistical analyses were performed with GraphPad Prism(GraphPad Inc). Differences between means (two samples) were assessed bythe Student's t test. Differences among multiple means were assessed byone-factor ANOVA followed by Tukey's multiple comparison test.

To validate the ability of the assay to quantitatively detect aggregatesthat bind thioflavin S, staining of BE-M17 cells overexpressing A53Tα-synuclein was carried out and the results revealed a rotenonedose-dependent increase in thioflavin S-positive aggregates relative tovehicle-treated control cells (FIGS. 4A-C). Higher magnification imagesobtained with a 40× objective indicated that the thioflavin S-positiveaggregates were intracellular and cytoplasmic, analogous to theaccumulation of intracytoplasmic Lewy bodies which are pathologicalhallmarks associated with Parkinson's disease (FIG. 4D). Quantitation ofthe area covered by thioflavin-S-positive aggregates established that 1and 5 μM rotenone were sufficient to induce robust aggregation (FIG. 4E)and thus are effective doses to test the ability of compounds toattenuate the formation of these aggregates.

Using the protocol described above, several compounds were selected andtested for their ability to reduce, prevent or eliminate thioflavinS-positive aggregates in rotenone-treated BE-M17 cells overexpressingA53T α-synuclein. Examples of results obtained from experiments usingthese compounds are described below.

In cells treated with 1 μM rotenone only, there was a robust presence ofthioflavin S-positive aggregates (FIG. 5A). Addition of 500 ng/ml or 1μg/ml compound 51 markedly reduced the abundance of theserotenone-induced aggregates by 87% and 91% respectively (FIGS. 5B-D;**p<0.01, ***p<0.001 relative to rotenone only-treated cells).Therefore, compound 51 reduced, prevented and/or eliminated thioflavinS-positive aggregates in cells that express human A53T α-synuclein.

Compound 76 was tested in a similar fashion. As expected, cells treatedwith 5 μM rotenone only, exhibited a robust presence of thioflavin.S-positive aggregates (FIG. 6A). Addition of 500 ng/ml, 1 μg/ml or 5μg/ml compound 76 markedly reduced the abundance of theserotenone-induced aggregates by 73%, 80% and 91% respectively (FIGS.6B-D; *p<0.05, **p<0.01 relative to rotenone only-treated cells).Therefore, compound 76 reduced, prevented and/or eliminated thioflavinS-positive aggregates in cells that express human A53T α-synuclein.

Likewise, compound 21 was tested using the thioflavin S aggregationassay. Cells treated with 5 μM rotenone only exhibited robust levels ofthioflavin S-positive aggregates (FIG. 7A), the abundance of which aremarkedly reduced by 84% through addition of 1 μg/ml compound 21 (FIGS.7B-C; *p<0.05 relative to rotenone only-treated cells). Therefore,compound 21 reduced, prevented and/or eliminated thioflavin S-positiveaggregates in cells that express human A53T α-synuclein.

Finally, compound 3 was also tested using the thioflavin S aggregationassay. As before, cells treated with 1 μM rotenone only exhibited robustlevels of thioflavin S-positive aggregates. Addition of 1 μg/ml or 5μg/ml compound 3 markedly reduced the abundance of theserotenone-induced aggregates by >90% (FIG. 8B-D; *p<0.05 relative torotenone only-treated cells). Therefore, compound 3 reduced, preventedand/or eliminated thioflavin S-positive aggregates in cells thatexpressed human A53T α-synuclein.

Taken together, we concluded that the tested compounds 51, 76, 21 and 3effectively and potently reduced, prevented, inhibited and/or eliminatedthe formation, deposition and/or accumulation of α-synuclein aggregatesin A53T α-synuclein-expressing BE-M17 cells.

Example 34 Compounds of This Invention Protect Against Rotenone-InducedCytotoxicity

The XTT Cytotoxicity Assay (Roche Diagnostics, Mannheim, Germany) waspreviously used to demonstrate that A53T α-synuclein potentiates celldeath in BE-M17 cells through an oxidative stress-dependent mechanism(Ostrerova-Golts et al., J. Neurosci., 20:6048-6054, 2000). Research hasshown that the accumulation of α-synuclein fibrils in Lewy bodiescontributes mechanistically to the degradation of neurons in Parkinson'sdisease and related disorders (Polymeropoulos et at, Science276:2045-2047, 1997; Kruger et al., Nature Genet. 18:106-108, 1998).Here, the XTT Cytotoxicity assay (hereafter referred to as the XTTassay) was used to measure the ability of test compounds to protectagainst rotenone-induced cytotoxicity (neuroprotective ability). Theassay is based on the principle that conversion of the yellowtetrazolium salt XTT to form an orange formazan dye (that absorbs lightaround 490 nm) occurs only in metabolically active, viable cells.Therefore, light absorbance at 490 nm is proportional to cell viability.For this assay, cells were plated in 96 well tissue culture dishes at10⁴ cells per well. After 24 hours, cells were treated with 500 nM or 2μM rotenone, or vehicle (0.05% DMSO) as indicated. Immediately afterrotenone addition, compounds were added at the indicated concentration.As a control, compounds were added without rotenone (vehicle only, 0.05%DMSO) and resulted in no toxicity at the doses tested. Untreated cellsreceived cell culture media only (no compound, with or withoutrotenone). After 40-44 hours of treatment, conditioned media was removedand replaced with 100 μl fresh media and 50 μl XTT labeling reactionmixture according to the manufacturer's recommendations. Four to fivehours later, the absorbance at 490 nm was measured and corrected forabsorbance at the 700 nm reference wavelength. Treatment with 500 nM and2 μM rotenone usually decreased viability by 30-40% and 40-50%respectively relative to untreated cell without rotenone. Percentinhibition of cell death was calculated as the proportion of therotenone-induced absorbance (viability) decrease that was eliminated bytest compound treatment.

In A53T α-synuclein-expressing BE-M17 cells treated with either 500 nMor 2 μM rotenone, there was a 30-50% loss of viability as expected.However, treatment with compound 51 at 50 μg/ml inhibited therotenone-induced loss of viability by 25-30% at both rotenone doses(FIG. 9). To better define the optimal dose of compound 51, theexperiment was repeated using 500 nM rotenone and 10 μg/ml or 25 μg/mlcompound 51 (data not shown). At these doses using compound 51 there wasa 45% and 55% inhibition of rotenone-induced toxicity, respectively,indicating that 25 μg/ml approximates the optimal therapeutic dose ofcompound 51 in this system.

The experiment was also performed with compound 76. Treatment with 5μg/ml of compound 76 inhibited the rotenone-induced loss of viability byapproximately 18% at both rotenone doses (FIG. 10). Similarly, treatmentwith compound 21 at 50 μg/ml inhibited the rotenone-induced loss ofviability by approximately 30-40% at both rotenone doses (FIG. 11).

Another compound, compound 63 showed a high degree of inhibition ofrotenone-induced loss of viability. For example, treatment with aslittle as 5 μg/ml resulted in some amount (10-13%) of inhibition,whereas treatment with 50 μg/ml resulted in approximately 55% and 35%inhibition of toxicity, first induce by 500 nM and 2 μM rotenone,respectively (FIG. 12).

In conclusion, the tested compounds were efficacious in inhibitingrotenone-induced cytotoxicity demonstrating neuroprotective activityagainst α-synuclein toxicity.

Example 35 Compounds of This Invention Directly Inhibit the In VitroConversion of α-synuclein to α-synuclein Fibrils Containing β-sheetSecondary Structure

As described above, thioflavin S histochemistry in α-synucleinexpressing cells was used to detect aggregates containing a high degreeof β-pleated structures that formed in response to rotenone treatment.Since several compounds were shown to reduce the abundance of thioflavinS-positive aggregates (Example 33), we sought independent confirmationthat the compounds directly inhibit the conversion of α-synuclein toβ-sheet containing structures by using circular dichroism (CD)spectroscopy. For this purpose, α-synuclein was obtained from rPeptide(Athens, Ga., USA) as a lyophilized salt in 1 mg aliquots. Buffercomponents and other solvents were obtained from Sigma as A.C.S. Reagentgrade or higher. Wild-type α-synuclein was dissolved in a buffercontaining 9.5 mM phosphate and 139.7 mM potassium chloride, adjusted topH 7.4. Solutions containing 1 mg/ml α-synuclein with the compounds (at1 mg/ml unless otherwise indicated) or without (vehicle, 0.95% ethanol)were agitated at 37 to induce aggregation. Aliquots of the solutionswere taken at the appropriate time point for CD spectral analysis. CDspectra were acquired on a Jasco J-810 spectropolarimeter using a 0.1 cmpath length cell. All spectra were recorded with a step size of 0.1 nm,a bandwidth of 1 nm, and an α-synuclein concentration of 0.1 mg/ml. Thespectra were trimmed at the shortest wavelength which still provided adynode voltage less than 600V. The trimmed spectra were then subjectedto a data processing routine beginning with noise reduction by Fouriertransform followed by subtraction of a blank spectrum (compound orvehicle only without α-synuclein). These blank corrected spectra werethen zeroed at 260 nm and the units converted from millidegrees tospecific ellipticity.

Percent β-sheet was determined from processed spectra by subtracting theellipticity minimum value at approximately 218 nm from the maximum valueobserved at approximately 197 nm and referencing to a scale normalizedto nearly fully folded and unfolded reference values, consistent withprevious reports (Ramirez-Alvarado et al., J. Mol. Biol., 273:898-912,1997; Andersen et al., J. Am. Chem. Sec., 121:9879-9880, 1999) The fullyfolded reference value was found by performing the described calculationon the spectrum of α-synuclein that was fibrillized for 144 hours(complete fibrilization), and assigning this difference the arbitraryvalue of 96% β-sheet. The unfolded reference was provided by thespectrum from the same sample at the initial time point (t=0) andascribing the difference found here the arbitrary value of 3% β-sheet.These percent β-sheet values were then used to provide the respectiverelative % inhibition induced by the compounds.

Since compound 51 is a potent inhibitor of thioflavin S-positiveaggregates in BE-M17 cells, we predicted that it would also inhibit theconversion of natively unfolded α-synuclein to a β-sheet-rich structure.First, in order to confirm that α-synuclein is indeed converted to aβ-sheet-rich structure and to establish the timing of this conversion inour system, an aliquot of the incubation mixture (without compounds) wassampled at various time points and CD spectra collected. At 24 hours ofincubation, CD analysis revealed a large abundance of a β-sheet-richstructure, indicated by the pronounced specific ellipticity minimum at218 nm and maximum at 197 nm (FIG. 13A; vehicle). However, when compound51 was included in the reaction mixture, and the incubation mixturesampled 24 hours later, there was reduction of the β-sheet secondarystructure of α-synuclein fibrils due to the absence of the minimum at218 nm. Instead, a spectrum characteristic of random coil was exhibited(FIG. 13A; compound 51). We conclude that compound 51 prevents theconversion of natively unfolded α-synuclein to a β-sheet-rich structure.

In order to test whether the inhibitory effect of compound 51 persistsover time, an incubation mixture in which compound 51 was added at thebeginning (t=0 hr) was sampled at subsequent time points (24, 48, 72 and144 hours). A control reaction contained α-synuclein without anycompound (vehicle). In the reaction where no compounds were added,α-synuclein progresses to a structure with approximately 90%β-sheet-rich structure (FIG. 13B; vehicle) whereas analysis of samplesfrom the reaction in which compound 51 was added at the beginning (t=0hr) reveals that β-sheet-rich structure does not significantly exceedbaseline levels (approximately 10%) even at 144 hours. Interestingly,when compound 51 is added 24 hours after the beginning of the incubation(t=24 hr), a time at which β-sheet-rich structure is approximately 30%,the conversion of α-synuclein to a β-sheet-rich structure is halted andfails to exceed approximately 45%, even after 144 hours at 37° C. (FIG.13B). Therefore, we concluded that compound 51 not only maintainsα-synuclein in its native unfolded state over time, but that compound 51also prevents further accumulation of a β-sheet-rich structure even whenthe conversion to such a structure has already begun.

To determine the necessary dose of compound 51 to inhibit conversion of1.0 mg/ml α-synuclein to a β-sheet-rich structure, a dose response curvewas generated using compound 51 at 0.01, 0.05, 0.10, 0.25, 0.50 and 1.0mg/ml (FIG. 13C). As little as 0.25 mg/ml (added at the beginning of thereaction) was sufficient to completely eliminate the conversion ofα-synuclein to a β-sheet-rich structure after 24 hours incubation at 37°C., and 0.15 mg/ml of compound 51 was sufficient to inhibit theconversion by approximately 50%.

Like compound 51, compound 76 also potently inhibited the accumulationof thioflavin S-positive aggregates in A53T-expressing α-synucleinBE-M17 cells. Thus, compounds 51 and 76, as well as compounds 63 and 23,were added individually to α-synuclein and the abundance of β-sheet-richstructure was measured after 48 hours in each reaction using CDspectroscopy. A control reaction in which vehicle only was added toα-synuclein showed 90% β-sheet-rich structure by 48 hours. As expected,compound 51 completed inhibited conversion of α-synuclein to aβ-sheet-rich structure, as did compound 76. Compounds 63 and 23,however, showed modest inhibitory effects on β-sheet formation.

Taken together, these results show that compounds 51 and 76, are potentand persistent inhibitors of α-synuclein aggregation, a hallmark of thesynucleinopathies such as Parkinson's disease. In addition compounds 23and 63 also show inhibition.

The present invention is not limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing descriptions. Such modificationsare intended to fall within the scope of the appended claims. Variouspublications are cited herein, the disclosures of which are incorporatedby reference in their entireties.

1. A method of treating the formation, deposition, accumulation, orpersistence of α-synuclein/NAC fibrils, comprising treating the fibrilswith an effective amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof, where: R is a C₃-C₁₀alkylene group, in which: a) there are optionally 1 or 2 non-adjacentdouble bonds; b) 2 non-adjacent methylene groups are replaced by NR′,where R′ is H or alkyl; and c) 1 or 2 methylene groups are replaced by acarbonyl group.
 2. The method of claim 1 wherein the compound isselected from:

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 1wherein treatment is provided to a mammal.
 4. The method of claim 3,wherein the mammal is a human.
 5. The method of claim 1, wherein theamount of the compound administered is between 0.1 mg/Kg/day and 1000mg/Kg/day.
 6. The method of claim 5, wherein the amount of compoundadministered is between 1 mg/Kg/day and 100 mg/Kg/day.
 7. The method ofclaim 5, wherein the amount of compound administered is between 10mg/Kg/day and 100 mg/Kg/day.
 8. A method of inhibiting or relieving asynuclein disease, comprising administration of a therapeuticallveffective amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof, where: R is a C₃-C₁₀alkylene group, in which: a) there are optionally 1 or 2 non-adjacentdouble bonds; b) 2 non-adjacent methylene groups are replaced by NR′,where R′ is H or alkyl; and c) 1 or 2 methylene groups are replaced by acarbonyl group.
 9. The method of claim 8, wherein the synuclein diseaseis selected from the group consisting of Parkinson's disease, familialParkinson's disease, Lewy body disease, the Lewy body variant ofAlzheimer's disease, dementia with Lewy bodies, multiple system atrophy,and the Parkinsonism-dementia complex of Guam.
 10. The method of claim8, wherein the synuclein disease is Parkinson's disease.
 11. The methodof claim 8, wherein the amount of the compound administered is between0.1 mg/Kg/day and 1000 mg/Kg/day.
 12. The method of claim 11, whereinthe amount of compound administered is between 1 mg/Kg/day and 100mg/Kg/day.
 13. The method of claim 11, wherein the amount of compoundadministered is between 10 mg/Kg/day and 100 mg/Kg/day.
 14. A method ofproviding neuroprotection from a synuclein disease to a mammalcomprising the step of administrating a therapeutically effective amountof a compound of the formula:

or a pharmaceutically acceptable salt thereof, where: R is a C₃-C₁₀alkylene group, in which: a) there are optionally 1 or 2 non-adjacentdouble bonds; b) 2 non-adjacent methylene groups are replaced by NR′,where R′ is H or alkyl; and c) 1 or 2 methylene groups are replaced by acarbonyl group.
 15. The method of claim 14, wherein the synucleindisease is selected from the group consisting of Parkinson's disease,familial Parkinson's disease, Lewy body disease, the Lewy body variantof Alzheimer's disease, dementia with Lewy bodies, multiple systematrophy, and the Parkinsonism-dementia complex of Guam.
 16. The methodof claim 14, wherein the synuclein disease is Parkinson's disease. 17.The method of claim 14, wherein the amount of the compound administeredis between 0.1 mg/Kg/day and 1000 mg/Kg/day.
 18. The method of claim 17,wherein the amount of compound administered is between 1 mg/Kg/day and100 mg/Kg/day.
 19. The method of claim 17, wherein the amount ofcompound administered is between 10 mg/Kg/day and 100 mg/Kg/day.
 20. Themethod of claim 14 wherein the therapeutically effective amount ofcompound administered is administered orally.